Cardiac-specific targeting-peptide (ctp), compositions, and uses thereof

ABSTRACT

Disclosed is a synthetic, non-naturally occurring 12-amino acid peptide, Cardiac Targeting Peptide, belonging to the larger class of cell penetrating peptides, for delivery of both diagnostic and potentially therapeutic agents to the heart. Also disclosed are subsequent generations of this Cardiac Targeting Peptide with improved, higher efficiency of cardiac uptake for CTP-mediated delivery of antiarrhythmics subjects for treating Atrial fibrillation or Ventricular fibrillation, CTP-mediated delivery of neuregulin-1β for treating systolic heart failure (SHF), and Szeto-Schiller peptides for treatment of diastolic congestive heart failure, among other applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/675,307, filed May 23, 2018; U.S. ProvisionalPatent Application No. 62/778,033, filed Dec. 11, 2018; and U.S.Provisional Patent Application No. 62/811,077, filed Feb. 27, 2019; thedisclosure of each of which is hereby incorporated by reference in itsentirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 21, 2019, isnamed UPB-013WO_ST25.txt and is 11,293 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to cardiac-specific targeting peptides(CTP)-mediated delivery of moieties having antiarrhythmic properties fortreating Atrial fibrillation or Ventricular fibrillation, andCTP-mediated delivery of neuregulin-1β for treating systolic heartfailure (SHF). The invention also relates generally to cardiac-specifictargeting peptide (CTP_(6aa)), compositions of CTP_(6aa), use of theCTP_(6aa) or a composition thereof in methods of treating cardiacconditions, and use of CTP_(6aa) in delivering molecular cargoesspecifically to cells of the heart.

BACKGROUND

Atrial fibrillation (also called AFib or AF) is a quivering or irregularheartbeat (arrhythmia) that can lead to blood clots, stroke, heartfailure and other heart-related complications. At least 2.7 millionAmericans are living with AFib. Ventricular fibrillation (VFib) is aserious cardiac disturbance that is a life-threatening rhythm and acommon cause of sudden cardiac death. It can be fatal. For many peoplewith this condition, irregular heart rhythms are the first and only signof coronary artery disease. VFib and AFib involve irregular heartrhythms, but they affect different parts of the heart. AFib can alsosignal a serious heart condition, but it is typically a symptom of achronic problem, not a life-threatening feature in itself (source:American Heart Association).

One of the most effective pharmaceutical therapies for AFib and VFib,amiodarone, has significant systemic toxicities leading to reducedlong-term utilization of the drug. The present disclosure addresses anunmet medical need for targeted delivery of cardiac therapeutics fortreating AFib and/or VFib by delivering therapeutic agents directly tocardiomyocytes, thereby reducing off-target effects and increasing theuse of existing, effective therapies.

A protein transduction peptide specific for the heart would be able todeliver biologic agents in a timely fashion to the heart when given atthe time of reperfusion for an infarction: Protein transduction domains(PTD) are small cationic peptides that can cross cellular membranes, andare able to transport large, biologically active molecules intomammalian cells in culture as well as in vivo. The limitation of PTDs isthe non-specific transduction of all tissue types with some tissues,such as liver and kidney, taking up the PTD much more avidly than hearttissue. Thus there is a need to identify peptides able to target cardiactissue specifically for delivery of biologics of therapeutic potential.

Although developments have been made to date, there is still an ongoingneed for new and effective peptides able to target cardiac tissuespecifically for delivery of biologics and/or cargo of therapeuticpotential.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a peptide comprising atwelve amino acid Cardiac-specific Targeting-Peptide (CTP_(12aa)) havinga sequence of Ala-Pro-Trp-His-Leu-Ser-Ser-Gln-Tyr-Ser-Arg-Thr (SEQ IDNO: 1), a six amino acid CTP (CTP_(6aa)) having a sequence of SQYSRT(SEQ ID NO: 5), or a twelve amino acid CTP having a sequence ofAAWHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A)) linked to a moiety havingantiarrhythmic properties (for example, amiodarone (IUPAC name:(2-butyl-1-benzofuran-3-yl)-[4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl]methanone;molecular formula: C₂₅H₂₉I₂NO₃)). In some embodiments, the moiety havingantiarrhythmic properties (for example, amiodarone) is linked upstreamof the N-terminus of the CTP (CTP_(12aa), CTP_(6aa), or CTP-P2A).

In one aspect, a peptide comprising a CTP of SEQ ID NO: 1, 2, 4, 5, 6,7, 8, 9, 10, 11, 28, 29, or 34 is linked to a moiety havingantiarrhythmic properties. Moieties with antiarrhythmic propertiesinclude compounds that can be grouped into 4 main classes ofantiarrhythmics (classes I, II, III, and IV) under the Vaughan Williamsclassification scheme. The Vaughan Williams classification schemecategorizes antiarrhythmics according to their dominant cellularelectrophysiologic effect and mechanism of action.

Class I compounds include sodium channel blockers (membrane-stabilizingdrugs) that can block fast sodium channels, slowing conduction infast-channel tissues (for example, in working atrial and ventricularmyocytes, and in the His-Purkinje system). Class I compounds can besubdivided into subclasses a, b, and c, with subclass a compounds (forexample, anticholinergics such as quinidine, procainamide, anddisopyramide) having moderate effects, subclass b compounds (forexample, lidocaine and lidocaine analogs, including tocainide andmexiletine) having weak effects, and subclass c compounds (for example,flecainide, propafenone, and moricizine) having strong effects.

Class II compounds include beta-blockers, which can affect predominantlyslow-channel tissues (for example, sinoatrial (SA) and atrioventricular(AV) node tissues), by decreasing rate of automaticity, slowingconduction velocity, and prolonging refractoriness. Examples of class IIcompounds include: carteolol, carvedilol, labetalol, nadolol,penbutolol, pindolol, propranolol, sotalol, timolol, acebutolol,atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and nebivolol.

Class III compounds include potassium channel blockers, which canprolong action potential duration and refractoriness in slow-channel andfast-channel tissues. Examples of class III compounds include:amiodarone, dronedarone, bretylium, sotalol, ibutilide, and dofetilide.

Class IV compounds include calcium channel blockers, for example, thedihydropyridine and non-dihydropyridine calcium channel blockers, thatcan depress calcium-dependent action potentials in slow-channel tissues,decreasing the rate of automaticity, slowing conduction velocity, andprolonging refractoriness.

Class IV dihydropyridine compounds include: amlodipine, felodipine,isradipine, nicardipine, nifedipine, nimodipine, and nitrendipine. ClassIV non-dihydropyridine compounds include: verapamil and diltiazem.

Moieties described herein having antiarrhythmic properties includeactive pharmaceutical compounds classified as class I antiarrhythmics,class II antiarrhythmics, class III antiarrhythmics, and class IVantiarrhythmics Exemplary antiarrhythmics conjugated to a CTP of thepresent disclosure include: amiodarone (synonyms: Amiodarona,Amiodaronum, Cordarone, Pacerone), flecainide (Tambocor), (Corvert),lidocaine (Xylocaine), procainamide (Procan, Procanbid), propafenone(Rythmol), quinidine, and tocainide (Tonocarid). For example, in someembodiments the disclosure provides a method of treating an atrialarrhythmia and/or a ventricular arrhythmia (for example, prematureventricular contractions, ventricular tachycardia, atrial fibrillation,or ventricular fibrillation), where the method includes administering apeptide comprising a CTP (for example, a CTP of SEQ ID NO: 1, 2, 4, 5,6, 7, 8, 9, 10, 11, 28, 29, or 34) linked to amiodarone to a patient,for example, a patient in need of treatment.

In certain embodiments a peptide comprising a CTP of SEQ ID NO: 1 islinked to a moiety having antiarrhythmic properties, for exampleamiodarone, and used in a method of treating cardiac tissue or a cardiaccondition in a subject in need thereof. In certain embodiments a peptidecomprising a CTP of SEQ ID NO: 2 is linked to a moiety havingantiarrhythmic properties, for example amiodarone, and used in a methodof treating cardiac tissue or a cardiac condition in a subject in needthereof. In certain embodiments a peptide comprising a CTP of SEQ ID NO:5 is linked to a moiety having antiarrhythmic properties, for exampleamiodarone, and used in a method of treating cardiac tissue or a cardiaccondition in a subject in need thereof. In certain embodiments, thecardiac condition is an atrial arrhythmia, for example, atrialfibrillation. In certain embodiments, the cardiac condition is aventricular arrhythmia, for example, ventricular tachycardia.

In one aspect, a peptide comprising a CTP of SEQ ID NO: 1, 2, 4, 5, 6,7, 8, 9, 10, 11, 28, 29, or 34 is linked to Neuregulin-1β, and used in amethod of treating cardiac tissue or a cardiac condition in a subject inneed thereof. In certain embodiments, the cardiac condition is systolicheart failure (SHF). Thus, in some embodiments the disclosure provides amethod of treating heart failure, for example, SHF, where the methodincludes administering a peptide comprising a CTP (for example, a CTP ofSEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 28, 29, or 34) linked toNeuregulin-1β to a patient, for example, a patient in need of treatment.

In one aspect the invention provides a peptide comprising a CTP_(6aa)comprising the sequence of Xaa₁ Xaa₂ Y Xaa₃ Xaa₄ T (SEQ ID NO: 4), inwhich Xaa₁, Xaa₂, Xaa₃, and Xaa₄ is any naturally occurring amino acid,linked to a moiety having antiarrhythmic properties, for exampleamiodarone, or to Neuregulin-1β. In some embodiments, a moiety havingantiarrhythmic properties or Neuregulin-1β is linked upstream of theN-terminus of the CTP_(6aa). In certain embodiments, Xaa₁ in theCTP_(6aa) SEQ ID NO: 4 is serine (S). In certain embodiments, Xaa₂ inthe CTP_(6aa) of SEQ ID NO: 4 is glutamine (Q). In certain embodiments,Xaa₃ in the CTP_(6aa) of SEQ ID NO: 4 is serine (S). In certainembodiments, Xaa₄ in the CTP_(6aa) of ID SEQ NO: 4 is arginine (R). Incertain embodiments, Xaa₁ and Xaa₂ in the CTP_(6aa) of SEQ ID NO: 4 areserine (S) and glutamine (Q), respectively. In certain embodiments, Xaa₁and Xaa₃ in the CTP_(6aa) of SEQ ID NO: 4 are both serine (S). Incertain embodiments, Xaa₁ and Xaa₄ in the CTP_(6aa) of SEQ ID NO: 4 areserine (S) and arginine (R), respectively. In certain embodiments, Xaa₂and Xaa₃ in the CTP_(6aa) of ID SEQ NO:4 are glutamine (Q) and serine(S), respectively. In certain embodiments, the CTP_(6aa) comprises thesequence SQYSRT (SEQ ID NO: 5). In certain embodiments, a peptidecomprising a CTP_(6aa) comprising the sequence of Xaa₁ Xaa₂ W Xaa₃ Xaa₄T (SEQ ID NO: 23), in which Xaa₁, Xaa₂, Xaa₃, and Xaa₄ is any naturallyoccurring amino acid, is linked to a moiety having antiarrhythmicproperties, for example amiodarone, or to Neuregulin-1β.

In one aspect the invention provides a peptide comprising a CTP_(6aa)comprising the sequence of S Q Xaa₁ S R Xaa₂ (SEQ ID NO: 6) linked to amoiety having antiarrhythmic properties, for example, amiodarone. Insome embodiments, a moiety having antiarrhythmic properties, for exampleamiodarone, is linked upstream of the N-terminus of the CTP_(6aa).

In one aspect the invention provides a peptide comprising a CTP_(6aa)comprising the sequence of S Q Xaa₁ S R Xaa₂ (SEQ ID NO: 6) linked toNeuregulin-1β. In some embodiments, Neuregulin-1β is linked upstream ofthe N-terminus of the CTP_(6aa).

In certain embodiments, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is alanine(A) and the CTP_(6aa) comprises the sequence of SQASRXaa₂ (SEQ ID NO:7), or optionally, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tryptophan(W) and the CTP_(6aa) comprises the sequence of SQWSRXaa₂ (SEQ ID NO:8), or Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tyrosine (Y) and theCTP_(6aa) comprises the sequence of SQYSRXaa₂ (SEQ ID NO: 9). In certainembodiments, Xaa₂ in the CTP_(6aa) of SEQ ID NO: 6 is threonine (T), andXaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is alanine (A), tryptophan (W), ortyrosine (Y) comprising the sequence of SQASRT (SEQ ID NO: 10), SQWSRT(SEQ ID NO: 11), or SQYSRT (SEQ ID NO: 5), respectively. In certainembodiments, Xaa₂ in the CTP_(6aa) of SEQ ID NO: 6 is alanine (A). Incertain embodiments, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tyrosine(Y) and Xaa₂ is alanine (A). In certain embodiments, the CTP_(6aa)comprises the sequence SQYSRT (SEQ ID NO: 5).

In certain embodiments, a peptide comprising a CTP_(6aa) of SEQ ID NO: 4and SEQ ID NO: 6, for example SEQ ID NO: 5, is a recombinant orsynthetically prepared peptide.

In some embodiments, a second peptide sequence, protein, or smallmolecule is linked upstream of the N-terminus of the CTP (CTP_(12aa),CTP_(6aa), or CTP-P2A). In some embodiments the linked peptide caninclude an ester linkage between the CTP and the second peptidesequence, protein, or small molecule, and the ester linkage can only becleaved by an intracellular esterase.

In some embodiments, a peptide comprising a CTP (CTP_(12aa), CTP_(6aa),or CTP-P2A) linked to a moiety having antiarrhythmic properties, forexample amiodarone, is optionally further labelled at both the C- andN-termini. For example, in some embodiments, the peptide is labelledwith a green fluorescent moiety, for example, 6-carboxyfluorosceine, anda red fluorescent moiety, for example, Cy5.5. For example, in someembodiments, the peptide is labelled with a green fluorescent moiety atits N-terminus and a red fluorescent moiety at its C-terminus. In someembodiments, the peptide is labelled with a red fluorescent moiety atits N-terminus and a green fluorescent moiety at its C-terminus.

It is contemplated that any of the foregoing CTP peptides of SEQ ID NOs:1-35 may, for example, be conjugated to a water-soluble polymer, e.g.,polyethylene glycol (PEG). In certain embodiments, the CTP_(6aa) of SEQID NO: 5 is conjugated to a water-soluble polymer, e.g., polyethyleneglycol (PEG).

In another aspect, the invention provides an isolated nucleic acidcomprising a nucleotide sequence encoding any one of the foregoing CTPpeptides of SEQ ID NOs: 1-35. In certain embodiments, the nucleotidesequence is codon optimized for expression in a host cell, e.g., anEscherichia coli cell. The invention also provides an expression vectorthat comprises any one of the foregoing nucleotide sequences. Similarly,the invention provides host cells, e.g., Escherichia coli cells,comprising one or more of the foregoing expression vectors. The peptidecan be produced in isolation in expression vectors or as a fusionpeptide/protein at the C-terminus of the peptide/protein to bedelivered, either with an intervening ester linkage or in continuity.

In another aspect, the invention provides a pharmaceutical compositioncomprising a peptide and any variants of any one of SEQ ID NOs: 1-35,and at least one pharmaceutically acceptable carrier and/or anexcipient. The peptide may be in a soluble form or in a crystal form.Furthermore, the composition may comprise a pH increasing agent. It iscontemplated that the pharmaceutical composition may, for example, beformulated as an oral dosage form or a parenteral dosage form. Incertain embodiments, the composition is formulated as a powder,granulate, pellet, micropellet, or a minitablet that can be orallyadministered, or as a lyophilized powder that can be reconstituted innormal saline or deiodized water for intravenous injection.

These and other aspects and features of the invention are described inthe following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D are bar graphs that quantify thefluorescence intensity of H9C2 cells transduced with cardiac-specifictargeting peptide alanine mutants labeled with Cy5.5. The sequence foreach indicated alanine mutant is provided in Table 2.

FIG. 2 is a series of bar graphs that show the transduction efficienciesand viability of various cell types incubated with Cy5.5 labeledcardiac-specific targeting peptide variants. FIG. 2A shows thetransduction of H9C2 cells in a first experiment. FIG. 2B shows thetransduction of H9C2 cells in a second experiment. FIG. 2C shows thetransduction of H9C2 cells and MEF cells. FIG. 2D shows the transductionof H9C2 and 3T3 cells. FIG. 2E shows the viability of H9C2 cells and MEFcells transduced with the Cy5.5 labeled cardiac-specific targetingpeptide variants. CTP (Full Length): NH₂-APWHLSSQYSRT-COOH (SEQ ID NO:1); CTP-A: NH₂-APWHLS-COOH (SEQ ID NO: 24); CTP-B: NH₂-SQYSRT-COOH(CTP_(6aa); SEQ ID NO: 5); cyclic: cyclized version of the indicatedpeptide; CTP-Random: random, linear, 12-amino acid peptide (RAN:NH₂-STLMKFCYVEQN-COOH ((SEQ ID NO: 26)); DMSO: Dimethyl sulfoxide.

FIG. 3 shows delivery of a rhodamine cargo to the intracellularcompartments of cardiomyocytes when the rhodamine cargo is conjugated atthe C-terminus of full-length CTP (e.g., CTP_(6aa)) Immunohistochemicalcharacterization of human iPSC derived beating cardiomyocytes transducedwith a dual-labeled carboxyfluorescein (green) and rhodamine (red)cardiac-specific targeting peptide was performed. iPSC derivedcardiomyocytes showed staining for troponin (FIG. 3A), calciumtransients (FIG. 3B), and robust delivery of the rhodamine cargo, whilethe carboxyfluorescein conjugated to the N-terminus of CTP appears to bereleased/secreted from the cells (FIG. 3C). FIG. 3D is a dark-fieldimage of the cell architecture. Scale bars represent 30 μm.

FIG. 4 is a series of bar graphs that show the transduction efficienciesof H9C2 cells incubated with Cy5.5 labeled cardiac-specific targetingpeptide variants. CTP (Full Length): NH₂-APWHLSSQYSRT-COOH (SEQ ID NO:1); CTP-A: NH₂-APWHLS-COOH (SEQ ID NO: 24); CTP-B: NH₂-SQYSRT-COOH(CTP_(6aa); SEQ ID NO: 5); cyclic: cyclized version of the peptide; RAN:a random, linear, 12-amino acid peptide (RAN: NH₂-STLMKFCYVEQN-COOH (SEQID NO: 26)); DMSO: Dimethyl sulfoxide.

FIG. 5 is a bar graph showing Cy5.5 intensity (arbitrary units (“AU”))in mouse myocardial tissue at various time points (15, 30, 45, 60, 90 or360 minutes) post-injection of CTP-Cy5.5 or a control random peptidesequence fused to Cy5.5 (RAN-Cy5.5).

FIG. 6A and FIG. 6B are bar graphs showing average fluorescent intensitymeasured in heart tissue injected with CTP constructs (CTP, CTP-B, H4A,or P2A) or random control peptide sequence (RAN).

DETAILED DESCRIPTION

The invention is based, in part, upon the unexpected discovery of a sixamino acid cardiac-specific targeting-peptide (CTP_(6aa)) with efficientcardiac-tissue specific transduction/cell penetration property, and, ineffect, efficient cardiac-tissue specific cargo-delivery and therapeuticproperties. The CTP_(6aa) of the present disclosure has fewer aminoacids than the full-length CTP described in U.S. Pat. No. 9,249,184,and, therefore, would be less immunogenic than the full-length CTP. Thepresent disclosure provides that the CTP_(6aa) of the present disclosurewould be advantageous, compared to the full-length CTP or an N-terminusfragment of the CTP, in delivering a cargo and/or a therapeutic agent tothe cardiac tissue of a mammalian subject (e.g., human).

Various features and aspects of the invention are discussed in moredetail below.

In certain embodiments, the present disclosure provides a CTP peptide ofTable 1 herein below, linked to a therapeutic moiety for treating acardiac disease and/or condition.

TABLE 1 Sequence of CTP_(6aa) of the SEQ ID NO: present disclosure  1APWHLSSQYSRT  2 AAWHLSSQYSRT (CTP-P2A)  3 Amiodarone-SQYSRT (CTP-B)  4Xaa₁ Xaa₂ Y Xaa₃ Xaa₄ T  5 SQYSRT (CTP-B)  6 S Q Xaa₁ S R Xaa₂  7SQASRXaa₂  8 SQWSRXaa₂  9 SQYSRXaa₂ 10 SQASRT 11 SQWSRT 12S Xaa₂ Y Xaa₃ Xaa₄ T 13 Xaa₁ Q Y Xaa₃ Xaa₄ T 14 Xaa₁ Xaa₂ Y S Xaa₄ T 15Xaa₁ Xaa₂ Y Xaa₃ R T 16 S Q Y Xaa₃ Xaa₄ T 17 S Xaa₂ Y S Xaa₄ T 18S Xaa₂ Y Xaa₃ R T 19 Xaa₁ Q Y S Xaa₄ T 20 S Xaa₂ Y S R T 21Xaa₁ Q Y S R T 22 S Q Y Xaa₃ R T 23 Xaa₁ Xaa₂ W Xaa₃ Xaa₄ T 24APWHLS (CTP-A) 25 Amiodarone-AAWHLSSQYSRT (CTP-P2A) 26 H4A APWALSSQYSRT27 L5A APWHASSQYSRT 28 S6A APWHLASQYSRT 29 S7A APWHLSAQYSRT 30 Q8AAPWHLSSAYSRT 31 Y9A APWHLSSQASRT 32 S10A APWHLSSQYART 33 R11AAPWHLSSQYSAT 34 T12A APWHLSSQYSRA 35 W3A APAHLSSQYSRT

The CTP of any one of SEQ ID NOs: 1-35 is a recombinant or syntheticallyprepared peptide.

I. Six Amino Acid Cardiac-Specific Targeting-Peptide (CTP_(6aa))

The present disclosure provides for Cardiac Targeting Peptides (“CTP”).In certain non-limiting embodiments the CTP specifically targets cardiactissue. “Specifically targets cardiac tissue” means that when said CTP,linked to a cargo molecule to form a CTP-cargo complex, is injected intoa mammal, the CTP-cargo complex is transduced into cardiac tissue atmuch higher levels than it is transduced into other tissues, such as,for example, liver, kidney, lung, skeletal muscle, or brain. In certainembodiments the ratio of transduction of a CTP that “specificallytargets cardiac tissue” into cardiac tissue relative to liver, kidney,lung, skeletal muscle or brain is at least 2:1 or is at least 3:1.

In one aspect the invention is based, in part, upon the discovery of asix amino acid Cardiac-specific Targeting-Peptide (CTP_(6aa)) comprisingthe sequence of Xaa₁ Xaa₂ Y Xaa₃ Xaa₄ T (SEQ ID NO: 4). In certainembodiments, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 4 is serine (S). Incertain embodiments, Xaa₂ in the CTP_(6aa) of SEQ ID NO: 4 is glutamine(Q). In certain embodiments, Xaa₃ in the CTP_(6aa) of SEQ ID NO: 4 isserine (S). In certain embodiments, Xaa₄ in the CTP_(6aa) of SEQ ID NO:1 is arginine (R). In certain embodiments, Xaa₁ and Xaa₂ in theCTP_(6aa) of SEQ ID NO: 4 are serine (S) and glutamine (Q),respectively. In certain embodiments, Xaa₁ and Xaa₃ in the CTP_(6aa) ofSEQ ID NO: 4 are both serine (S). In certain embodiments, Xaa₁ and Xaa₄in the CTP_(6aa) of SEQ ID NO: 4 are serine (S) and arginine (R),respectively. In certain embodiments, Xaa₂ and Xaa₃ in the CTP_(6aa) ofSEQ ID NO: 4 are glutamine (Q) and serine (S), respectively. In certainembodiments, the CTP_(6aa) comprises the sequence SQYSRT (SEQ ID NO: 5).

In one aspect the invention is based, in part, upon the discovery of asix amino acid Cardiac-specific Targeting-Peptide (CTP_(6aa)) comprisingthe sequence of S Q Xaa₁ S R Xaa₂ (SEQ ID NO: 6). In certainembodiments, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is alanine (A) andthe CTP_(6aa) comprises the sequence of SQASRXaa₂ (SEQ ID NO: 7), oroptionally, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tryptophan (W) andthe CTP_(6aa) comprises the sequence of SQWSRXaa₂ (SEQ ID NO: 8), orXaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tyrosine (Y) and the CTP_(6aa)comprises the sequence of SQYSRXaa₂ (SEQ ID NO: 8). In certainembodiments, Xaa₂ in the CTP_(6aa) of SEQ ID NO: 6 is threonine (T), andXaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is alanine (A), tryptophan (W), ortyrosine (Y) comprising the sequence of SQASRT (SEQ ID NO: 10), SQWSRT(SEQ ID NO: 11), or SQYSRT (SEQ ID NO: 5), respectively. In certainembodiments, Xaa₂ in the CTP_(6aa) of SEQ ID NO: 6 is alanine (A). Incertain embodiments, Xaa₁ in the CTP_(6aa) of SEQ ID NO: 6 is tyrosine(Y) and Xaa₂ is alanine (A). In certain embodiments, the CTP_(6aa)comprises the sequence SQYSRT (SEQ ID NO: 5).

In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4 and SEQ ID NO: 6,for example SEQ ID NO: 5, is a recombinant or synthetically preparedpeptide.

In one aspect, CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 isconjugated to a nanoparticle. In certain embodiments the CTP_(6aa) ofSEQ ID NO: 5 is conjugated to a nanoparticle.

In certain non-limiting embodiments the CTP_(6aa) has a net charge ofbetween about +0.8 to +1.2 at pH=7. In certain non-limiting embodimentsthe CTP_(6aa) peptide has a net charge of about 1.1 at pH=7.

In certain non-limiting embodiments, the CTP_(6aa) has an isoelectricpoint at between pH 8 and pH 9. In certain non-limiting embodiments, theCTP_(6aa) peptide has an isoelectric point at pH 8.56.

In certain non-limiting embodiments, the CTP_(6aa) has an averagehydrophilicity index of between −0.2 and −0.6. In certain non-limitingembodiments, the CTP_(6aa) peptide has an average hydrophilicity indexof −0.4.

In certain non-limiting embodiments, the CTP of any one of SEQ ID NOs:1-35 is comprised of (L) amino acids.

In certain non-limiting embodiments, the CTP of any one of SEQ ID NOs:1-35 is comprised of (D) amino acids.

In certain embodiments, any of the foregoing CTP peptides of any one ofSEQ ID NOs: 1-35 may be isolated. For example, in certain embodiments,the CTP_(6aa) of SEQ ID NO: 1, 2, or 5 is isolated.

In another aspect, the invention provides CTP_(6aa) comprising at leastone (for example, one or two) mutation(s) at a position corresponding towild type CTP_(6aa) of SEQ ID NO: 5, wherein the at least one mutationis present at position 3 or 6. In certain embodiments, the mutation maybe a conservative substitution relative to wild type CTP_(6aa) of SEQ IDNO: 5, whereas in certain other embodiments, the mutation may benon-conservative substitutions relative to wild type CTP_(6aa) of SEQ IDNO: 5.

As used herein, the term “conservative substitution” refers to asubstitution with a structurally similar amino acid. Non conservativesubstitutions are amino acid substitutions that are not conservativesubstitutions.

As disclosed herein, a CTP_(6aa) of any one of SEQ ID NOs: 5-9, forexample SEQ ID NO: 5, has higher transducing/penetrating activity thanthat of SEQ ID NO: 1 or 24. For example, a CTP_(6aa) of SEQ ID NO: 5 or6 may have from 2-50 fold higher transducing activity than that of SEQID NO: 1 or 24. In certain embodiments, the CTP_(6aa) of any one of SEQID NOs: 5-9, for example SEQ ID NO: 5, has from about 2 to about 50,from about 2 to about 40, from about 2 to about 30, from about 2 toabout 20, from about 2 to about 10, from about 2 to about 5, from about5 to about 50, from about 5 to about 40, from about 5 to about 30, fromabout 5 to about 20, from about 5 to about 10, from about 10 to about50, from about 10 to about 40, from about 10 to about 30, from about 10to about 20, from about 20 to about 50, from about 20 to about 40, fromabout 20 to about 30, from about 30 to about 50, from about 30 to about40, from about 40 to about 50, about 2, about 5, about 10, about 20,about 30, about 40, or about 50 fold higher transducing activity thanthat of SEQ ID NO: 1 or 2.

It is contemplated that a disclosed CTP of any one of SEQ ID NOs: 1-35,for example SEQ ID NO: 1, 2, or 5, may be modified, engineered orchemically conjugated. For example, it is contemplated that a disclosedCTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or 5,can be conjugated to an effector agent using standard in vitroconjugation chemistries. If the effector agent is another polypeptide,the CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or5, can be chemically conjugated to the effector or joined to theeffector as a fusion protein. Construction of fusion proteins is withinordinary skill in the art.

In certain embodiments, depending upon a particular mode ofadministration or site of activity, a disclosed CTP of any one of SEQ IDNOs: 1-35, for example SEQ ID NO: 1, 2, or 5, can be modified with amoiety that improves its stabilization and/or retention in circulation,e.g., in blood, serum, or other tissues. For example, a disclosed CTP ofany one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or 5, may beconjugated to a polymer, e.g., a substantially non-antigenic polymer,such as a polyalkylene oxide or a polyethylene oxide. In certainembodiments, a disclosed CTP of any one of SEQ ID NOs: 1-35, for exampleSEQ ID NO: 1, 2, or 5, is conjugated to a water-soluble polymer, e.g., ahydrophilic polyvinyl polymer, e.g., polyvinylalcohol orpolyvinylpyrrolidone. Examples of such polymers include polyalkyleneoxide homopolymers such as polyethylene glycol (PEG) or polypropyleneglycols, polyoxyethylenated polyols, copolymers thereof and blockcopolymers thereof. Additional useful polymers include polyoxyalkylenessuch as polyoxyethylene, polyoxypropylene, and block copolymers ofpolyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, andbranched or unbranched polysaccharides.

In another aspect, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or11 is conjugated to a detectable agent. In certain embodiments, theCTP_(6aa) of SEQ ID NO: 5 is conjugated to a detectable agent. Incertain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10,or 11 is conjugated to a detectable agent via an ester linkage.

In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is conjugated to a radionuclide or radioactive moiety, biotin,luciferase, an enzyme, rhodamine, a fluorophore, nanoparticle,microbubbles, liposomes or a luminescent moiety. In certain embodiments,the CTP_(6aa) of SEQ ID NO: 5 is conjugated to a radionuclide orradioactive moiety, biotin, luciferase, an enzyme, rhodamine, afluorophore, nanoparticle, microbubbles, liposomes or a luminescentmoiety. In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8,5, 9, 10, or 11 is conjugated to a radionuclide or radioactive moiety,biotin, luciferase, an enzyme, rhodamine, a fluorophore, nanoparticle,microbubbles, liposomes or a luminescent moiety, via an ester linkage.

In another aspect, the present disclosure provides a method of imagingor detecting a tissue, comprising administering an effective amount of acomposition comprising the detectable agent-conjugated CTP_(6aa) of SEQID NO: 4, 6, 7, 8, 5, 9, 10, or 11 to a subject; exposing the subject toconditions conducive to detection of the CTP_(6aa) conjugate; andobtaining an image. In certain embodiments, the detectableagent-conjugated CTP_(6aa) of SEQ ID NO: 5 is used in a method ofimaging or detecting a tissue.

In another aspect, the present disclosure provides a CTP_(6aa) of SEQ IDNO: 4, 6, 7, 8, 5, 9, 10, or 11 formulated as a delivery vehicle/agent.In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is conjugated to a drug or therapeutic, a nanoparticle, apeptide, a protein, or a detectable agent. In certain embodiments, theCTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 is conjugated to adrug or therapeutic, a nanoparticle, a peptide, a protein, or adetectable agent via an ester linkage. In certain embodiments, theCTP_(6aa) of SEQ ID NO: 5 is formulated as a delivery vehicle/agent.

In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is linked/conjugated to the drug or therapeutic, thenanoparticle, the peptide, the protein, or the detectable agent via anester linkage, disulfide or protease sensitive linkers. In certainembodiments, the CTP_(6aa) of SEQ ID NO: 5 is linked/conjugated to thedrug or therapeutic, the nanoparticle, the peptide, the protein, or thedetectable agent via an ester linkage, disulfide, or protease sensitivelinkers.

In certain embodiments, the nanoparticle conjugated to the CTP_(6aa) ofSEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 comprises a drug or therapeuticagent selected from an antibody or fragment thereof, SB239063,superoxide dismutase, HGF, FGF-1, FGF-2, an NF-κB inhibitor, NSDpeptide, heme oxygenase, an antioxidant, iNOS, S100A1, catalase,glutathione peroxidase, a TGFβ inhibitor, VEGF, sonic hedgehog protein,HGF, an IAP, prostaglandin, Pyrvinium Pamoate, and Diprotin A,Szeto-Schiller peptide, cyclosporine, amiodarone.

In another aspect, the present disclosure provides a method of treatingcardiac tissue or a cardiac condition in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a composition comprising the CTP_(6aa) of SEQ ID NO: 4, 6, 7,8, 5, 9, 10, or 11. In certain embodiments, the CTP_(6aa) of SEQ ID NO:5 is used in a method of treating cardiac tissue or a cardiac conditionin a subject in need thereof.

In another aspect, the present disclosure provides a method ofintroducing a cargo into a cardiac muscle cell comprising administering,to the cardiac muscle cell, an amount of a complex comprising theCTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 linked to a cargoeffective to introduce the cargo into the muscle cell. In certainembodiments, the cargo comprises a radioisotope, fluorescent marker,gadolinium marker, luciferase marker, microsphere or nanoparticle. Incertain embodiments, the CTP_(6aa) of SEQ ID NO: 5 is used in a methodof introducing a cargo into a cardiac muscle cell. In certainembodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 isconjugated to the cargo via an ester linkage.

In another aspect, the present disclosure provides a method of treatinga human subject suffering from a myocardial infarction, comprisingintroducing a cargo into a cardiac muscle cell of the human subjectcomprising administering, to the human subject, a therapeuticallyeffective amount of a complex comprising the CTP_(6aa) of SEQ ID NO: 4,6, 7, 8, 5, 9, 10, or 11 linked to a cargo, where the cargo inhibitscell death, inhibits arrhythmias, improves contractility, lengthenssubject survival, or a combination thereof. In certain embodiment, thecargo is selected from an NF-κB inhibitor, NSD peptide, heme oxygenase,an antioxidant, iNOS, S100A1, superoxide dismutase, catalase,glutathione peroxidase, a TGFβ inhibitor, VEGF, FGF-1, FGF-2, sonichedgehog protein, HGF and an IAP. In certain embodiments, the CTP_(6aa)of SEQ ID NO: 5 is used in a method of treating a human subjectsuffering from a myocardial infarction. In certain embodiments, theCTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 is conjugated to thecargo via an ester linkage.

In another aspect, the present disclosure provides a method of treatinga human subject suffering from a myocardial infarction, comprisingintroducing a cargo into a cardiac muscle cell of the human subjectcomprising administering, to the human subject, a therapeuticallyeffective amount of a complex comprising the CTP_(6aa) of SEQ ID NO: 4,6, 7, 8, 5, 9, 10, or 11 linked to a cargo, wherein the cargo inhibitscell death, inhibits arrhythmias, improves contractility, lengthenssubject survival, or a combination thereof. In certain embodiments, thecargo is selected from an NF-κB inhibitor, NBD peptide, heme oxygenase,an antioxidant, iNOS, S100A1, superoxide dismutase, catalase,glutathione peroxidase, a TGFβ inhibitor, VEGF, FGF-1, FGF-2, sonichedgehog protein, HGF and an IAP. In certain embodiments, the CTP_(6aa)of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11 is linked/conjugated to thecargo via an ester linkage.

In another aspect, the present disclosure provides a method of treatinga subject suffering from a metabolic defect that damages the heartcomprising introducing a cargo into a cardiac muscle cell of the humansubject comprising administering, to the subject, a therapeuticallyeffective amount of a complex comprising the CTP_(6aa) of SEQ ID NO: 4,6, 7, 8, 5, 9, 10, or 11 linked to a cargo, wherein the cargo correctsthe metabolic defect. In certain embodiments, the metabolic defect isGaucher's disease and the cargo is glucocerebrosidase. In certainembodiments, the CTP_(6aa) of SEQ ID NO: 5 is used in a method oftreating a subject suffering from a metabolic defect that damages theheart. In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8,5, 9, 10, or 11 is linked/conjugated to the cargo via an ester linkage.

In another aspect, the present disclosure provides a method of treatinga subject suffering from a metabolic defect that damages the heartcomprising introducing a cargo into a cardiac muscle cell of the humansubject comprising administering, to the subject, a therapeuticallyeffective amount of a complex comprising the CTP_(6aa) of SEQ ID NO: 4,6, 7, 8, 5, 9, 10, or 11 linked to a cargo, where the cargo corrects themetabolic defect. In certain embodiments, the metabolic defect isGaucher's disease and the cargo is glucocerebrosidase. In certainembodiments, the CTP_(6aa) of SEQ ID NO: 5 is used in a method oftreating a subject suffering from a metabolic defect that damages theheart. In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8,5, 9, 10, or 11 is linked/conjugated to the cargo via an ester linkage.

In another aspect, the present disclosure provides a method ofintroducing a detectable cargo into a cardiac muscle cell comprisingadministering, to the cardiac muscle cell, an amount of a complexcomprising the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11linked to a cargo effective to introduce the cargo into the muscle cell,wherein the cargo comprises a detectable compound. In certainembodiments, the detectable compound comprises a detectableradioisotope, fluorescent marker, gadolinium marker, or luciferasemarker. In certain embodiments, the CTP_(6aa) of SEQ ID NO: 5 is used ina method of introducing a detectable cargo into a cardiac muscle cell.In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is linked/conjugated to the cargo via an ester linkage.

In another aspect, the present disclosure provides a method ofintroducing a detectable cargo into a cardiac muscle cell comprisingadministering, to the cardiac muscle cell, an amount of a complexcomprising the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11linked to a cargo effective to introduce the cargo into the muscle cell,wherein the cargo comprises a detectable compound. In certainembodiments, the detectable compound comprises a detectableradioisotope, fluorescent marker, gadolinium marker, or luciferasemarker. In certain embodiments, the CTP_(6aa) of SEQ ID NO: 5 is used ina method of introducing a detectable cargo into a cardiac muscle cell.In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is conjugated to the cargo via an ester linkage.

In another aspect, the invention provides a CTP_(6aa) of any one of SEQID NOs: 5-9, for example SEQ ID NO: 5, having a higher level of cardiactransduction compared to the transduction of a peptide of SEQ ID NO: 1or SEQ ID NO: 24, for example, under the conditions set forth in Example1.

It is contemplated that any of the foregoing CTP_(6aa) peptides of SEQID NOs: 4-11 may, for example, have 5-20 fold higher transducingcapacity, compared to a peptide of SEQ ID NO: 1 or SEQ ID NO: 24.

II. Cargo

According to the invention, the CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 5, is linked with a cargo molecule to form a complex,optionally via a linker molecule or molecules. The cargo molecule may bea protein (including a glycoprotein), a nucleic acid, a carbohydrate, alipid, or a combination thereof. In some embodiments, the cargo moleculeis an active pharmaceutical ingredient, for example, an activepharmaceutical ingredient with antiarrhythmic properties. For example,in some embodiments described herein, a cargo molecule is a class Iantiarrhythmic compound (for example, a subclass a compound, a subclassb compound, or a subclass c compound), a class II antiarrhythmiccompound, a class III antiarrhythmic compound, a class IV antiarrhythmiccompound, a sodium channel blocker, an anticholinergic compound,quinidine, ajmaline, hydroquinidine, lorajmine, prajmaline, sparteine,procainamide, disopyramide, lidocaine, a lidocaine analog, phenytoin,tocainide, mexiletine, flecainide, indecainide, lorcainide, encainide,ethacizine, propafenone, moracizine, a beta-blocker, carteolol,carvedilol, labetalol, nadolol, penbutolol, pindolol, propranolol,sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, esmolol,metoprolol, nebivolol, a potassium channel blocker, amiodarone,dronedarone, E-4031, vernakalant, nifekalant, bretylium, bunaftine,celivarone, sotalol, ibutilide, dofetilide, tedisamil, a calcium channelblocker, a dihydropyridine calcium channel blocker, anon-dihydropyridine calcium channel blocker, amlodipine, felodipine,isradipine, nicardipine, nifedipine, nimodipine, nitrendipine,verapamil, diltiazem, adenosine, a benzodiazepine, a barbiturate, amuscarinic antagonist, a muscarinic agonist, digoxin, digitoxin,ouabain, magnesium sulfate, or atropine.

In certain non-limiting embodiments, the cargo is a protein. In certainnon-limiting embodiments, the protein is selected from the groupconsisting of a cytokine, a growth factor, an enzyme, an ion channel,and an anti-inflammatory protein.

In certain non-limiting embodiments, the cargo is an antioxidant.

In certain non-limiting embodiments, the cargo is a nucleic acid.Non-limiting examples of such nucleic acid include DNA, RNA, antisenseRNA, interfering RNA, microRNA, catalytic RNA, and catalytic DNA.

The present disclosure provides Cardiac Targeting Peptides (“CTP”)linked/conjugated to Neuregulin-1β-CTP or a moiety having antiarrhythmicproperties, for example amiodarone. In certain non-limiting embodiments,the N-terminus of a CTP_(12aa) comprising the sequence of APWHLSSQYSRT(SEQ ID NO: 1), a CTP_(6aa) having a sequence of SQYSRT (SEQ ID NO: 5),or a CTP_(12aa) having a sequence of AAWHLSSQYSRT (SEQ ID NO: 2(CTP-P2A)) is linked/conjugated to Neuregulin-1β-CTP or a moiety havingantiarrhythmic properties, for example amiodarone. In certainembodiments, the CTP is linked/conjugated to a second a moiety havingantiarrhythmic properties, for example amiodarone.

In certain non-limiting embodiments, the CTP linked to a therapeuticmoiety specifically targets cardiac tissue. “Specifically targetscardiac tissue” means that when said CTP conjugate, linked to a secondpeptide sequence, a protein, or a small molecule to form a conjugatedprotein, is injected into a mammal, the conjugated protein is transducedinto cardiac tissue at much higher levels than it is transduced intoother tissues, such as, for example, liver, kidney, lung, skeletalmuscle, or brain.

In certain non-limiting embodiments, the CTP (e.g., CTP_(6aa) of SEQ IDNO: 5) has an average hydrophilicity index of between −0.2 and −0.6. Incertain non-limiting embodiments, the CTP (e.g., CTP of SEQ ID NO: 5)has an average hydrophilicity index of −0.4.

In certain non-limiting embodiments, the CTP of SEQ NO: 1, 2, 3, 4, or 5is comprised of (L) amino acids. In certain non-limiting embodiments,the CTP of SEQ ID NO: 1, 2, 3, 4, or 5 is comprised of (D) amino acids.

The present disclosure is based, in part, on directly deliveringNeuregulin-1β or a moiety having antiarrhythmic properties, for exampleamiodarone, to the heart using CTP to target peptide delivery to cellsof the heart, for example, cardiomyocytes. For example, in someembodiments the invention provides a method of delivering Neuregulin-1βor a moiety having antiarrhythmic properties, for example, amiodarone,to cells of a subject's heart, for example, a subject's cardiomyocytes.In some embodiments, the subject is a mammal, for example, a primate,for example a human. Thus, in some embodiments the invention provides amethod of delivering Neuregulin-1β or a moiety having antiarrhythmicproperties, for example, amiodarone, to cells of a human subject'sheart, for example, a human subject's cardiomyocytes. In someembodiments the invention provides a method of delivering Neuregulin-1βor a moiety having antiarrhythmic properties, for example, amiodarone,to cells of a subject's heart, for example, a subject's cardiomyocytes,by administering a peptide that includes CTP conjugated toantiarrhythmics, for example, by Schiff base chemistry, to the subject.In some embodiments, the peptide can be introduced by means of a cell ora virus that includes a nucleic acid encoding a CTP sequence.

In certain embodiments, the invention provides formulating a peptidecomposition, for example, an antiarrhythmic moiety-CTP composition orNeuregulin-1β-CTP composition, in a stable formulation for delivery tocells in vitro as well as to animals via intravenous perfusion. Incertain embodiments, the invention provides delivering the formulatedcomposition to a mammalian cardiomyoblast cell and monitoring forcellular toxicity by performing a cell viability assay. In certainembodiments, the invention provides delivering a peptide, for example,an antiarrhythmic moiety-CTP or Neuregulin-1β-CTP, to a mammaliancardiomyoblast cell line. In certain embodiments, the invention providesdelivering an antiarrhythmic moiety-CTP to a mammal having arrhythmias,for example atrial and/or ventricular fibrillation. In some embodiments,the invention also includes observing one or more echocardiographicparameters, for example, left ventricular wall thickness, leftventricular function, and/or mitral in-flow patterns, to assessdiastolic relaxation at various time points before, during, and/or afterdelivery and/or treatment. In certain embodiments, the inventionprovides administering an antiarrhythmic moiety-CTP or Neuregulin-1β-CTPintravenously weekly, for example, for 6-12 weeks, and assessing forhepatic, renal, CNS, and/or cardiac toxicity using blood chemistryand/or histology.

In certain embodiments, the invention provides formulating a peptidecomposition that includes CTP linked/conjugated to a second moietyhaving an antiarrhythmic property, in a stable formulation for deliveryto cells in vitro as well as to animals via intravenous perfusion. Incertain embodiments, the invention provides delivering the formulatedcomposition to a mammalian cardiomyoblast cell and monitoring forcellular toxicity by performing a cell viability assay. In certainembodiments, the invention provides delivering a peptide that includesCTP linked/conjugated to a second moiety having an antiarrhythmicproperty, to a mammalian cardiomyoblast cell line. In certainembodiments, the disclosure provides a method of administering a peptidethat includes CTP linked/conjugated to a second moiety havingantiarrhythmic properties, where the administering is performedintravenously on a weekly basis for a designated period of time, forexample, for 6-12 weeks. In some embodiments, the method furtherincludes assessing for hepatic, renal, CNS, and/or cardiac toxicityusing blood chemistry and/or histology.

In another aspect the present disclosure also provides sustained releaseformulations (long-lasting formulation) suitable for injection todeliver a peptide comprising Cardiac-specific Targeting-Peptide (CTP)linked/conjugated to Neuregulin-1β or a moiety having antiarrhythmicproperties.

In another aspect, the present disclosure provides a method of imagingor detecting a heart tissue (e.g., a human heart tissue), byadministering a peptide that includes CTP linked/conjugated to a moietyhaving an antiarrhythmic property to a subject; exposing the subject toconditions conducive to detection of the peptide; and obtaining animage.

In embodiments, the invention provides a peptide that includes a twelveamino acid Cardiac-specific Targeting-Peptide (CTP_(12aa)) comprisingthe sequence of Ala-Pro-Trp-His-Leu-Ser-Ser-Gln-Tyr-Ser-Arg-Thr (SEQ IDNO: 1) linked/conjugated to a second peptide sequence, a protein, or asmall molecule that has reactive oxygen species (ROS) scavengingproperties. In some embodiments, the second peptide sequence, protein,or small molecule is conjugated upstream of the N-terminus of the CTPpeptide. In some embodiments the peptide can include an ester linkagebetween the CTP and the second peptide sequence, protein, or smallmolecule, and the ester linkage can only be cleaved by an intracellularesterase. Examples of peptide sequences, proteins, and small moleculeswith ROS scavenging properties include, but are not limited to,heme-oxygenase 1, resveratrol, N-acetyl-cysteine,N-tert-butyl-α-phenylnitrone, and4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.

In some embodiments, the peptide is optionally further labelled at boththe C- and N-termini. For example, in some embodiments, the peptide islabelled with a green fluorescent moiety, for example,6-carboxyfluorescein, and a red fluorescent moiety, for example, Cy5.5.For example, in some embodiments, the peptide is labelled with a greenfluorescent moiety at its N-terminus and a red fluorescent moiety at itsC-terminus. In some embodiments, the peptide is labelled with a redfluorescent moiety at its N-terminus and a green fluorescent moiety atits C-terminus.

The present disclosure is based, in part, on directly delivering an ROSscavenging peptide (for example, SS-31 or SS-02), protein, or smallmolecule to the heart using CTP to target peptide delivery to cells ofthe heart, for example, cardiomyocytes. ROS scavenging peptides includeSzeto-Schiller (SS) peptides (for example, SS-01, SS-02, and SS-31),which contain alternating aromatic and basic amino acids, includingtyrosine or dimethyltyrosine, and which are highly cell permeable. SSpeptides are described in Szeto HH, (2006) “Cell-permeable,Mitochondrial-targeted, Peptide Antioxidants” The AAPS Journal8(2):E277-E283, the entire contents of which are incorporated herein byreference. In some embodiments the invention provides a method ofdelivering an ROS scavenging peptide (for example, SS-31 or SS-02),protein, or small molecule to cells of a subject's heart, for example, asubject's cardiomyocytes. In some embodiments, the subject is a mammal,for example, a primate, for example, a human. Thus, in some embodimentsthe invention provides a method of delivering an ROS scavenging peptide(for example, SS-31 or SS-02), protein, or small molecule to cells of ahuman subject's heart, for example, a human subject's cardiomyocytes. Insome embodiments the invention provides a method of delivering an ROSscavenging peptide (for example, SS-31 or SS-02), protein, or smallmolecule to cells of a subject's heart, for example, a subject'scardiomyocytes, by administering a peptide that includes CTPlinked/conjugated to an ROS scavenging peptide (for example, SS-31 orSS-02), protein, or small molecule, for example, by an ester linkage, tothe subject. In some embodiments, the peptide can be introduced by meansof a cell or a virus that includes a nucleic acid encoding a CTPlinked/conjugated peptide sequence.

In some aspects, the invention provides a method of scavenging ROS incell culture by exposing a cell culture (for example, cells and/or cellculture media) to a peptide that includes CTP linked/conjugated to asecond peptide sequence, a protein, or a small molecule with ROSscavenging properties. For example, in some embodiments, the inventionprovides a method of scavenging ROS in cell culture by exposing a cellculture to a peptide that includes CTP linked/conjugated to SS-31(SS-CTP-31) or CTP conjugated to SS-02 (SS-CTP-02). In some embodiments,exposing a cell culture to a peptide that includes CTP linked/conjugatedto a second peptide sequence, a protein, or a small molecule with ROSscavenging properties is achieved by expressing the peptide in one ormore cells in the cell culture and/or by viral infection.

In certain embodiments, the invention provides formulating a peptidecomposition, for example, an SS-CTP composition, in a stable formulationfor delivery to cells in vitro as well as to animals via intravenousperfusion. In certain embodiments, the invention provides delivering theformulated composition to a mammalian cardiomyoblast cell and monitoringfor cellular toxicity by performing a cell viability assay. In certainembodiments, the invention provides delivering a peptide, for example,an SS-CTP, to a mammalian cardiomyoblast cell line challenged withoxidative stress (for example, a cardiomyoblast cell line challengedwith oxidative stress using hydrogen peroxide) as a stressor. In someembodiments, the invention also includes assessing mitochondrialfunction at baseline, under stress with H₂O₂, and in cells treated withan SS-CTP prior to challenge with H₂O₂ using a Seahorse analyzer(Agilent Technologies, Inc., Santa Clara, Calif., USA). In certainembodiments, the invention provides delivering an SS-CTP to a mammalhaving diastolic heart failure (DHF). In some embodiments, the inventionalso includes observing one or more echocardiographic parameters, forexample, left ventricular wall thickness, left ventricular function,and/or mitral in-flow patterns, to assess diastolic relaxation atvarious time points before, during, and/or after delivery and/ortreatment. In certain embodiments, the invention provides administeringan SS-CTP intravenously weekly, for example, for 6-12 weeks, andassessing for hepatic, renal, CNS, and/or cardiac toxicity using bloodchemistry and/or histology.

In certain embodiments, the invention provides formulating a peptidecomposition that includes CTP linked/conjugated to a second peptidesequence, a protein, or a small molecule with ROS scavenging properties,in a stable formulation for delivery to cells in vitro as well as toanimals via intravenous perfusion. In certain embodiments, the inventionprovides delivering the formulated composition to a mammaliancardiomyoblast cell and monitoring for cellular toxicity by performing acell viability assay. In certain embodiments, the invention providesdelivering a peptide that includes CTP linked/conjugated to a secondpeptide sequence, a protein, or a small molecule with ROS scavengingproperties, to a mammalian cardiomyoblast cell line challenged withoxidative stress (for example, a cardiomyoblast cell line challengedwith oxidative stress using hydrogen peroxide) as a stressor. In someembodiments, the invention also includes assessing mitochondrialfunction at baseline, under stress with H₂O₂, and in cells treated withthe peptide prior to challenge with H₂O₂, using Seahorse. In certainembodiments, the invention provides delivering a peptide that includesCTP linked/conjugated to a second peptide sequence, a protein, or asmall molecule with ROS scavenging properties to a mammal havingdiastolic heart failure (DHF). In some embodiments, the invention alsoincludes observing one or more echocardiographic parameters, forexample, left ventricular wall thickness, left ventricular function,and/or mitral in-flow patterns, to assess diastolic relaxation atvarious time points before, during, and/or after delivery and/ortreatment. In certain embodiments, the invention provides administeringa peptide that includes CTP linked/conjugated to a second peptidesequence, a protein, or a small molecule with ROS scavenging propertiesintravenously weekly, for example, for 6-12 weeks, and assessing forhepatic, renal, CNS, and/or cardiac toxicity using blood chemistryand/or histology.

In another aspect, the present disclosure provides a method of imagingor detecting a heart tissue (e.g., a human heart tissue), byadministering a peptide that includes CTP linked/conjugated to a secondpeptide sequence, a protein, or a small molecule with ROS scavengingproperties (for example, an SS-CTP conjugate) to a subject; exposing thesubject to conditions conducive to detection of the peptide, andobtaining an image.

In certain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9,10, or 11 is linked/conjugated or fused to a tafazzin peptide. Incertain embodiments, the CTP_(6aa) of SEQ ID NO: 4, 6, 7, 8, 5, 9, 10,or 11 is linked/conjugated or fused to the tafazzin peptide through apolypeptide linker. In certain embodiments, the CTP_(6aa) of SEQ ID NO:5 is conjugated or fused to a tafazzin peptide.

In certain embodiments, one specific non-limiting example of a cargo isa NF-κB inhibitor, for example NBD peptide TALDWSWLQTE (SEQ ID NO:35).One specific non-limiting example of a cargo is heme oxygenase, forexample human heme oxygenase. One specific non-limiting example of acargo is inducible nitric oxide synthase (“iNOS”), for example humaniNOS.

In certain embodiments, one specific non-limiting example of a cargo isS100A1 (an inotropic regulator of myocardial contractility. One specificnon-limiting example of a cargo is extracellular superoxide dismutase.Further specific non-limiting examples of cargo include Cu/Zn-SOD,Mn-SOD, catalase, and glutathione peroxidase. One specific non-limitingexample of cargo is transforming growth factor beta (“TGFβ”) type IIreceptor (Ad.CAG-s TGFβII), a competitive inhibitor of TGFβ. Onespecific non-limiting example of cargo is VEGF (vascular endothelialgrowth factor), for example human VEGF. One specific non-limitingexample of cargo is fibroblast growth factor (FGF), for example humanFGF-1 or FGF-2. One specific non-limiting example of cargo is hepatocytegrowth factor (“HGF”). One set of non-limiting examples of cargo is anapoptosis inhibitor, such as one of the so-called inhibitors ofapoptosis (“IAPs”), for example, the human IAPs c-IAP1, c-IAP2, andXIAP. One specific non-limiting example of cargo is Sonic Hedgehogprotein. One specific non-limiting example of cargo isglucocerebrosidase, for example human glucocerebrosidase used fortreatment in Gaucher's disease. One specific non-limiting example ofcargo is an RNAi that inhibits expression of TGFβ.

In another set of non-limiting embodiments, the cargo is a nanoparticleor a microsphere containing a diagnostic or therapeutic agent. Inanother set of non-limiting embodiments, the cargo is a vectorcomprising a therapeutic gene, for example an adenovirus vector or alentivirus vector. In another set of non-limiting embodiments, the cargois a detectable compound for analysis of uptake in viable cardiac cellsversus non-viable cells following ischemic injury. Non-limiting examplesof detectable compounds include fluorodeoxyglucose, a technetium-99 orother radioisotope-labelled cargo, fluorescent markers, gadoliniummarkers, etc. One non-limiting example of a radioisotope-labelled cargois Sestamibi, a coordination complex of the radioisotope technetium-99mwith the ligand methoxyisobutylisonitrile (“MIBI”).

III. Linkers

The CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or5, and the cargo are linked covalently or non-covalently, optionally viaone or more linker molecules. Where the bond is a covalent bond, CTP ofany one of SEQ ID NOs: 1-35 and cargo, optionally with a linker(s)between, may be joined via one or more peptide bond, thioester bond,thioether bond, carbamate bond, etc., which can be created according tomethods generally and well known in the art.

In certain embodiments, the linker may comprise a cleavage site thatmay, upon enzymatic or chemical cleavage, release the CTP of any one ofSEQ ID NOs: 1-35, for example SEQ ID NO: 5, from its cargo. In certainnon-limiting embodiments, the linker may be a ligand pair. As onespecific example, the linker may be an avidin/biotin pair.

Accordingly, the invention provides for a complex comprising a CTP ofany one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or 5, linkedto a cargo. The complex may comprise additional elements. For example,the CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or5, and/or cargo may be conjugated to one or more additional moleculethat improves delivery or stability. As one non-limiting example, theCTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or 5,and/or cargo may be PEGylated. As another non-limiting example, thecargo may be linked to a nuclear transport peptide. As anothernon-limiting example, the cargo may be linked to a detectable compound.

IV. Method of Introducing a Cargo

In another aspect, the present disclosure provides a method ofintroducing a cargo into a cardiac tissue comprising administering, tothe cardiac tissue, a complex comprising a CTP of any one of SEQ ID NOs:1-35, for example SEQ ID NO: 1, 2, or 5, linked to the cargo molecule.

The present disclosure provides for a method of introducing a cargo intoa cardiac muscle cell comprising administering, to the cardiac musclecell, a complex comprising a CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 1, 2, or 5, linked to the cargo. For example, thepresent disclosure provides a method of introducing a cargo into acardiac muscle cell comprising administering, to the cardiac musclecell, an amount of a complex comprising the CTP of any one of SEQ IDNOs: 1-35, for example SEQ ID NO: 1, 2, or 5, linked to a cargoeffective to introduce the cargo into the muscle cell. In certainembodiments, the cargo comprises a radioisotope, fluorescent marker,gadolinium marker, luciferase marker, microsphere or nanoparticle.

The present disclosure provides for a method of selectively delivering acargo to cardiac tissue in a subject, comprising administering, to thesubject, a complex comprising the CTP of any one of SEQ ID NOs: 1-35,for example SEQ ID NO: 1, 2, or 5, linked to the cargo. The presentdisclosure provides for a method of selectively delivering a cargo to acardiac muscle cell in a subject, comprising administering, to thesubject, a complex comprising a CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 1, 2, or 5, linked to the cargo.

In another aspect, the present disclosure provides a method ofintroducing a detectable cargo into a cardiac muscle cell comprisingadministering, to the cardiac muscle cell, an amount of a complexcomprising a CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO:5, linked to a cargo effective to introduce the cargo into the musclecell, wherein the cargo comprises a detectable compound. In certainembodiments, the detectable compound comprises a detectableradioisotope, fluorescent marker, gadolinium marker, or luciferasemarker.

V. Methods of Use

In one aspect, the present disclosure provides a method of treatingcardiac tissue or a cardiac condition in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a composition comprising a CTP of any one of SEQ ID NOs: 1-35,for example SEQ ID NO: 1, 2, or 5, linked to a therapeutic agent fortreating a cardiac disease and/or condition.

In certain embodiments, the present disclosure provides a method ofintroducing a moiety having antiarrhythmic properties into a cardiacmuscle cell comprising administering to the cardiac muscle cell aCTP_(12aa) comprising the sequence of APWHLSSQYSRT (SEQ ID NO: 1), aCTP_(6aa) having a sequence of SQYSRT (SEQ ID NO: 5), or a CTP_(12aa)having a sequence of AAWHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A)) conjugated toa moiety having antiarrhythmic properties, for example amiodarone, in anamount effective to introduce the moiety having antiarrhythmicproperties into the muscle cell. In certain embodiments, the peptidefurther comprises a microsphere or nanoparticle.

In certain embodiments, the present disclosure provides a method oftreating a human subject suffering from atrial and/or ventriculararrhythmias, comprising introducing a cargo into a cardiac muscle cellof the human subject by administering to the human subject a CTP_(12aa)comprising the sequence of APWHLSSQYSRT (SEQ ID NO: 1), a CTP_(6aa)having a sequence of SQYSRT (SEQ ID NO: 5), or a CTP_(12aa) having asequence of AAWHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A)) conjugated to a moietyhaving antiarrhythmic properties, for example amiodarone. In certainembodiments, the present disclosure provides a method of treatingventricular arrhythmias selected from premature ventricularcontractions, ventricular tachycardia, and ventricular fibrillation, byadministering to the human subject a CTP_(12aa) comprising the sequenceof APWHLSSQYSRT (SEQ ID NO: 1), a CTP_(6aa) having a sequence of SQYSRT(SEQ ID NO: 5), or a CTP_(12aa) having a sequence of AAWHLSSQYSRT (SEQID NO: 2 (CTP-P2A)) conjugated to a moiety having antiarrhythmicproperties, for example amiodarone.

In certain embodiments, the present disclosure provides a method oftreating a human subject suffering from SHF, comprising introducing acargo into a cardiac muscle cell of the human subject by administeringto the human subject a CTP_(12aa) comprising the sequence ofAPWHLSSQYSRT (SEQ ID NO: 1), a CTP_(6aa) having a sequence of SQYSRT(SEQ ID NO: 5), or a CTP_(12aa) having a sequence of AAWHLSSQYSRT (SEQID NO: 2 (CTP-P2A)) conjugated to Neuregulin-1β.

In certain embodiments, the present disclosure provides a method ofperforming myocardial perfusion analysis of a subject, by administeringto the subject a conjugate of the CTP comprising, for example, SEQ IDNO: 1, 2, 4, or 5 (e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP),and monitoring through Planar or SPECT imaging, delivery of theconjugate to the subject's heart.

In certain embodiments, the present disclosure provides a method ofimaging or detecting a heart tissue, by administering to the subject aconjugate of the CTP comprising, for example, SEQ ID NO: 1 (e.g., anantiarrhythmic-CTP or Neuregulin-1β-CTP); exposing the subject toconditions conducive to detection of the CTP conjugate; and obtaining animage. In this aspect, the antiarrhythmic-CTP optionally furthercomprises detectable moiety. In certain embodiments, theantiarrhythmic-CTP further comprises a detectable moiety.

Where a conjugate of the CTP comprising, for example, SEQ ID NO: 1(e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP) is administered to asubject, the subject may be a human subject or a non-human subject, suchas a primate, a companion animal (e.g., dog, cat, horse), a laboratoryanimal (e.g., mouse, rat, rabbit or guinea pig) or a farm animal (cow,goat, etc.).

A conjugate of the CTP comprising, for example, SEQ ID NO: 1, 2, 4, or 5(e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP) may be administeredby any route including but not limited to intravenous, intraatrial,intraperitoneal, subcutaneous, oral, rectal, etc.

The present disclosure provides for pharmaceutical compositionscomprising a CTP comprising, for example, SEQ ID NO: 1, 2, 4, or 5linked to a therapeutic agent (e.g., an antiarrhythmic-CTP orNeuregulin-1β-CTP), and a suitable pharmaceutical carrier, for example,water or physiologic saline.

The CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 1, 2, or5, disclosed herein can be used to treat various diseases or disordersassociated with cardiac tissue or muscle in a subject. In certainembodiments, the method comprises orally administering to the subject aneffective amount of a disclosed CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 1, 2, or 5, either alone or in a combination withanother therapeutic agent to treat the disease or disorder in thesubject.

In certain embodiments, the CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 1, 2, or 5, is conjugated or fused to a tafazzinpeptide. In certain embodiments, the CTP of any one of SEQ ID NOs: 1-35,for example SEQ ID NO: 1, 2, or 5, is conjugated or fused to thetafazzin peptide through a polypeptide linker.

In another aspect, the present disclosure provides a method of treatinga subject having a disorder associated with a tafazzin deficiency or aremodeled cardiolipin deficiency, comprising administering to thesubject a therapeutically effective amount of a composition comprisingthe tafazzin-conjugated CTP of any one of SEQ ID NOs: 1-35, for exampleSEQ ID NO: 1, 2, or 5. In certain embodiments, the tafazzin-conjugatedCTP_(6aa) of SEQ ID NO: 5 is used in treating a subject having adisorder associated with a tafazzin-deficiency or a remodeledcardiolipin-deficiency.

In another aspect, the present disclosure provides a method of treatinga subject having, or at risk of developing Barth syndrome, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising the tafazzin-conjugated CTP of any one of SEQ IDNOs: 1-35, for example SEQ ID NO: 5.

In certain embodiments, the present disclosure provides a method oftreating a subject having, or at risk of developing Barth syndrome,comprising administering to the subject a therapeutically effectiveamount of a composition comprising the tafazzin-conjugated CTP_(6aa) ofSEQ ID NO: 4, 6, 7, 8, 5, 9, 10, or 11. In certain embodiments, thetafazzin-conjugated CTP_(6aa) of SEQ ID NO: 5 is used for treating asubject having, or at risk of developing Barth syndrome.

In one aspect, the present disclosure provides a method of treatingcardiac tissue or a cardiac condition in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a composition comprising the nanoparticle-conjugated CTP ofany one of SEQ ID NOs: 1-35 linked to a moiety having antiarrhythmicproperties, for example amiodarone. In certain embodiments the CTP ofSEQ ID NO: 1, 2, or 5 is used for treating a cardiac tissue or a cardiaccondition in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a composition comprisingthe nanoparticle-conjugated CTP of SEQ ID NO: 1, 2, or 5 linked to amoiety having antiarrhythmic properties, for example amiodarone.

In another aspect, the present disclosure provides a method of treatinga human subject suffering from a myocardial infarction, comprisingintroducing a cargo into a cardiac muscle cell of the human subjectcomprising administering, to the human subject, a therapeuticallyeffective amount of a complex comprising the CTP of any one of SEQ IDNOs: 1-35, for example SEQ ID NO: 5, linked to a cargo, where the cargoinhibits cell death, inhibits arrhythmias, improves contractility,lengthens subject survival, or a combination thereof. In certainembodiments, the cargo is selected from an NF-κB inhibitor, NSD peptide,heme oxygenase, an antioxidant, iNOS, S100A1, superoxide dismutase,catalase, glutathione peroxidase, a TGFβ inhibitor, VEGF, FGF-1, FGF-2,sonic hedgehog protein, HGF and an IAP.

In another aspect, the present disclosure provides a method of treatinga subject suffering from a metabolic defect that damages the heartcomprising introducing a cargo into a cardiac muscle cell of the humansubject comprising administering, to the subject, a therapeuticallyeffective amount of a complex comprising the CTP of any one of SEQ IDNOs: 1-35, for example SEQ ID NO: 5, linked to a cargo, wherein thecargo corrects the metabolic defect. In certain embodiments, themetabolic defect is Gaucher's disease and the cargo isglucocerebrosidase.

The present disclosure provides for a method of treating a subjectsuffering from a myocardial infarction, comprising administering, to thesubject, a therapeutically effective amount of a complex comprising aCTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 5, linked tothe cargo, where the cargo (meaning the biological function of thecargo) inhibits cell death, inhibits arrhythmias, improvescontractility, lengthens subject survival, or a combination thereof.

The present disclosure provides for a method of treating a subjectsuffering from angina, comprising administering, to the subject, atherapeutically effective amount of a complex comprising a CTP of anyone of SEQ ID NOs: 1-35, for example SEQ ID NO: 5, linked to a cargo,where the cargo reduces the risk of myocardial infarction, inhibits celldeath, limits arrhythmias, improves contractility, lengthens subjectsurvival, or a combination thereof.

Non-limiting examples of cargo that may be used to treat myocardialinfarction and/or angina include an NF-κB inhibitor, NBD peptide, hemeoxygenase, an antioxidant, iNOS, S100A1, superoxide dismutase, catalase,glutathione peroxidase, a TGFβ inhibitor, VEGF, sonic hedgehog protein,FGF-1, FGF-2, HGF, and IAPs.

The present disclosure provides for a method of treating a subjectsuffering from a metabolic defect that damages the heart comprisingadministering, to the subject, a therapeutically effective amount of acomplex comprising a CTP of any one of SEQ ID NOs: 1-35, for example SEQID NO: 5, linked to a cargo, where the cargo corrects the metabolicdefect.

The present disclosure provides for a method of treating a subjectsuffering from Gaucher's disease comprising administering, to thesubject, a therapeutically effective amount of a complex comprising aCTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 5, linked toa glucocerebrosidase.

Where the CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO:5/cargo complex is administered to a subject, the subject may be a humansubject or a non-human subject, such as a primate, a companion animal(e.g., dog, cat, horse) a laboratory animal (e.g., mouse, rat, rabbit orguinea pig) or a farm animal (cow, goat, etc.).

The CTP of any one of SEQ ID NOs: 1-35, for example SEQ ID NO: 5-cargocomplex may be administered by any route including but not limited tointravenous, intraarterial, intraperitoneal, subcutaneous, oral, rectal,etc.

The present disclosure provides for pharmaceutical compositionscomprising CTP of any one of SEQ ID NOs: 1-35, for example, SEQ ID NO:5-cargo and a suitable pharmaceutical carrier, for example, water,physiologic saline.

In one aspect, the present disclosure provides a method of treating ahuman subject suffering from atrial and/or ventricular arrhythmias,comprising introducing a cargo into a cardiac muscle cell of the humansubject by administering to the human subject a CTP_(12aa) comprisingthe sequence of APWHLSSQYSRT (SEQ ID NO: 1), a CTP_(6aa) having asequence of SQYSRT (SEQ ID NO: 5), or a CTP_(12aa) having a sequence ofAAWHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A)) conjugated to a moiety havingantiarrhythmic properties, for example amiodarone. In certainembodiments, the present disclosure provides a method of treatingventricular arrhythmias selected from premature ventricularcontractions, ventricular tachycardia, and ventricular fibrillation, byadministering to the human subject a CTP_(12aa) comprising the sequenceof APWHLSSQYSRT (SEQ ID NO: 1), a CTP_(6aa) having a sequence of SQYSRT(SEQ ID NO: 5), or a CTP_(12aa) having a sequence of AAWHLSSQYSRT (SEQID NO: 2 (CTP-P2A)) conjugated to a moiety having antiarrhythmicproperties, for example amiodarone.

In one aspect, the present disclosure provides a method of treating ahuman subject suffering from SHF, comprising introducing a cargo into acardiac muscle cell of the human subject by administering to the humansubject a CTP_(12aa) comprising the sequence of APWHLSSQYSRT (SEQ ID NO:1), a CTP_(6aa) having a sequence of SQYSRT (SEQ ID NO: 5), or aCTP_(12aa) having a sequence of AAWHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A))conjugated to Neuregulin-1β.

In one aspect, the present disclosure provides a method of performingmyocardial perfusion analysis of a subject, by administering to thesubject a conjugate of the CTP comprising, for example, SEQ ID NO: 1, 2,4, or 5 (e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP), andmonitoring through Planar or SPECT imaging, delivery of the conjugate tothe subject's heart.

In another aspect, the present disclosure provides a method of imagingor detecting a heart tissue, by administering to the subject a conjugateof the CTP comprising, for example, SEQ ID NO: 1 (e.g., anantiarrhythmic-CTP or Neuregulin-1β-CTP); exposing the subject toconditions conducive to detection of the CTP conjugate; and obtaining animage. In this aspect, the antiarrhythmic-CTP optionally furthercomprises a detectable moiety.

Where a conjugate of the CTP comprising, for example, SEQ ID NO: 1(e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP) is administered to asubject, the subject may be a human subject or a non-human subject, suchas a primate, a companion animal (e.g., dog, cat, horse) a laboratoryanimal (e.g., mouse, rat, rabbit or guinea pig) or a farm animal (cow,goat, etc.).

A conjugate of the CTP comprising, for example, SEQ ID NO: 1, 2, 4, or 5(e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP) may be administeredby any route including but not limited to intravenous, intraatrial,intraperitoneal, subcutaneous, oral, rectal, etc.

The present disclosure provides for pharmaceutical compositionscomprising a conjugate of the CTP comprising, for example, SEQ ID NO: 1,2, 4, or 5 (e.g., an antiarrhythmic-CTP or Neuregulin-1β-CTP) and asuitable pharmaceutical carrier, for example, water or physiologicsaline.

VI. Pharmaceutical Compositions

For therapeutic use, a CTP of any one of SEQ ID NOs: 1-35, for exampleSEQ ID NO: 5, linked to a therapeutic agent (for example, amiodarone)described herein preferably is combined with a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” as usedherein refers to those compounds, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The term “pharmaceutically acceptable carrier” as used herein refers tobuffers, carriers, and excipients suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable carriers include any of the standard pharmaceutical carriers,such as a phosphate buffered saline solution, water, emulsions (e.g.,such as an oil/water or water/oil emulsions), and various types ofwetting agents. The compositions also can include stabilizers andpreservatives. For examples of carriers, stabilizers and adjuvants, see,e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ.Co., Easton, Pa. [1975]. Pharmaceutically acceptable carriers includebuffers, solvents, dispersion media, coatings, isotonic and absorptiondelaying agents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art.

In certain embodiments, the CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 5, linked to a therapeutic agent (for example,amiodarone) can be formulated, or co-administered (either at the sametime or sequentially), for example, by an enteral route (e.g., orally),with a pH increasing agent, for example, a protein pump inhibitor (PPI),to enhance the stability of the CTP of any one of SEQ ID NOs: 1-35, forexample SEQ ID NO: 5, for example, in an acidic environment, forexample, in the gastrointestinal tract.

Proton pump inhibitors are a group of drugs whose main action ispronounced and long-lasting reduction of gastric acid production. Protonpump inhibitors act by blocking the hydrogen/potassium adenosinetriphosphatase enzyme system (the H⁺/K⁺ ATPase, or more commonly justgastric proton pump) of the gastric parietal cell. The proton pump isthe terminal stage in gastric acid secretion, being directly responsiblefor secreting H⁺ ions into the gastric lumen, making it an ideal targetfor inhibiting acid secretion. Examples of proton pump inhibitorsinclude: Omeprazole (brand names: LOSEC®, PRILOSEC®, ZEGERID®);Lansoprazole (brand names: PREVACID®, ZOTON®, INHIBITOL®); Esomeprazole(brand names: NEXIUM®); and Pantoprazole (brand names: PROTONIX®,SOMAC®, PANTOLOC®).

Pharmaceutical compositions containing a CTP of any one of SEQ ID NOs:1-35, for example SEQ ID NO: 5, linked to a therapeutic agent (forexample, amiodarone) disclosed herein can be presented in a dosage unitform and can be prepared by any suitable method. A pharmaceuticalcomposition should be formulated to be compatible with its intendedroute of administration. The pharmaceutical compositions may be in avariety of forms. These include, for example, liquid, semi-solid andsolid dosage forms, such as liquid solutions (e.g., injectable andinfusible solutions), dispersions or suspensions, tablets, pills,powders, liposomes and suppositories. The preferred form will dependupon the intended mode of administration and therapeutic application.

Although the compositions preferably are formulated for administrationenterally (for example, orally), such compositions can be administeredby a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal,or intramuscular injection). The phrases “parenteral administration” and“administered parenterally” as used herein mean modes of administrationother than enteral and topical administration, usually by injection, andinclude, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andinfrasternal injection and infusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying that yield a powder of an agentdescribed herein plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The proper fluidity of asolution can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption ofinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin.

Depending upon the mode of administration, for example, by parenteraladministration, it may be desirable to produce a pharmaceuticalformulation that is sterile. Sterilization can be accomplished by anysuitable method, e.g., filtration through sterile filtration membranes.Where the composition is lyophilized, filter sterilization can beconducted prior to or following lyophilization and reconstitution.

In certain embodiments, a disclosed composition comprises a polyionicreagent which may, e.g., coat the CTP of any one of SEQ ID NOs: 1-35,for example SEQ ID NO: 5, linked to a therapeutic agent (for example,amiodarone), i.e., the composition comprises a polyionic coating.Exemplary polyionic reagents include PSS (poly(Sodium4-styrenesulfonate), PAA (poly Acrylic acid sodium salt), PMG(poly(methylene-co-guanidine) hydrochloride), DS (dextran sulfate), PMA(poly(methyl acrylate)), or PVS (polyvinylsiloxane).

Lyophilized Formulation

The lyophilized formulation for use in a method of treatment of thepresent disclosure includes the Cardiac-specific Targeting-Peptide (CTP)(e.g., any one of SEQ ID NOs: 1-35) linked to a drug or therapeutic(e.g., amiodarone) and a lyoprotectant. In certain embodiments, thelyophilized formulation for use in a method of treatment of the presentdisclosure includes the CTP_(12aa) of SEQ ID NO: 1 linked to a drug ortherapeutic (e.g., amiodarone) and a lyoprotectant. In certainembodiments, the lyophilized formulation for use in a method oftreatment of the present disclosure includes the CTP_(12aa) of SEQ IDNO: 2 linked to a drug or therapeutic (e.g., amiodarone) and alyoprotectant. In certain embodiments, the lyophilized formulation foruse in a method of treatment of the present disclosure includes theCTP_(6aa) of SEQ ID NO: 5 linked to a drug or therapeutic (e.g.,amiodarone) and a lyoprotectant. In certain embodiments, thelyoprotectant may be sugar, e.g., disaccharides. In certain embodiments,the lyoprotectant may be sucrose or maltose. In certain embodiments, thelyophilized formulation may also include one or more of a bufferingagent, a surfactant, a bulking agent, and/or a preservative.

The amount of sucrose or maltose useful for stabilization of thelyophilized drug product may be in a weight ratio of at least 1:2protein to sucrose or maltose. In certain embodiments, the protein tosucrose or maltose weight ratio may be of from 1:2 to 1:5.

In certain embodiments, the pH of the formulation, prior tolyophilization, may be set by addition of a pharmaceutically acceptableacid and/or base. In certain embodiments the pharmaceutically acceptableacid may be hydrochloric acid. In certain embodiments, thepharmaceutically acceptable base may be sodium hydroxide.

Before lyophilization, the pH of the solution containing the protein ofthe present disclosure may be adjusted between about 6 to about 8. Incertain embodiments, the pH range for the lyophilized drug product maybe from about 7 to about 8.

In certain embodiments, a salt or buffer components may be added in anamount of about 10 mM-about 200 mM. The salts and/or buffers arepharmaceutically acceptable and are derived from various known acids(inorganic and organic) with “base forming” metals or amines. In certainembodiments, the buffer may be phosphate buffer. In certain embodiments,the buffer may be glycinate, carbonate, citrate buffers, in which case,sodium, potassium or ammonium ions can serve as counterion.

In certain embodiments, a “bulking agent” may be added. A “bulkingagent” is a compound which adds mass to a lyophilized mixture andcontributes to the physical structure of the lyophilized cake (e.g.,facilitates the production of an essentially uniform lyophilized cakewhich maintains an open pore structure). Illustrative bulking agentsinclude mannitol, glycine, polyethylene glycol and sorbitol. Thelyophilized formulations of the present disclosure may contain suchbulking agents.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

In certain embodiments, the lyophilized drug product for use in a methodof treatment of the present disclosure may be constituted with anaqueous carrier. The aqueous carrier of interest herein is one which ispharmaceutically acceptable (e.g., safe and non-toxic for administrationto a human) and is useful for the preparation of a liquid formulation,after lyophilization. Illustrative diluents include sterile water forinjection (SWFI), bacteriostatic water for injection (BWFI), a pHbuffered solution (e.g. phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution.

In certain embodiments, the lyophilized drug product of the currentdisclosure is reconstituted with either Sterile Water for Injection, USP(SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution,the lyophilized powder dissolves into a solution.

In certain embodiments, the lyophilized protein product of the instantdisclosure is constituted to about 4.5 mL water for injection anddiluted with 0.9% saline solution (sodium chloride solution).

Liquid Formulation

In embodiments, the Cardiac-specific Targeting-Peptide (CTP) (e.g., anyone of SEQ ID NOs: 1-35) linked to a drug or therapeutic (e.g.,amiodarone) of the present disclosure is formulated as a liquidformulation for use in a method of treatment described herein. Theliquid formulation may be presented at a 10 mg/mL concentration ineither a USP/Ph Eur type I 50R vial closed with a rubber stopper andsealed with an aluminum crimp seal closure. The stopper may be made ofelastomer complying with USP and Ph Eur. In certain embodiments vialsmay be filled with an effective amount of the CTP conjugated to a drugor therapeutic solution in order to allow an extractable volume. Incertain embodiments, the liquid formulation may be diluted with 0.9%saline solution.

In certain embodiments, the liquid formulation for use in a method ofthe disclosure may be prepared as a solution in combination with a sugarat stabilizing levels. In certain embodiments the liquid formulation maybe prepared in an aqueous carrier. In certain embodiments, a stabilizermay be added in an amount no greater than that which may result in aviscosity undesirable or unsuitable for intravenous administration. Incertain embodiments, the sugar may be disaccharides, e.g., sucrose. Incertain embodiments, the liquid formulation may also include one or moreof a buffering agent, a surfactant, and a preservative.

In certain embodiments, the pH of the liquid formulation may be set byaddition of a pharmaceutically acceptable acid and/or base. In certainembodiments, the pharmaceutically acceptable acid may be hydrochloricacid. In certain embodiments, the base may be sodium hydroxide.

In addition to aggregation, deamidation is a common product variant ofpeptides and proteins that may occur during fermentation, harvest/cellclarification, purification, drug substance/drug product storage andduring sample analysis. Deamidation is the loss of NH₃ from a proteinforming a succinimide intermediate that can undergo hydrolysis. Thesuccinimide intermediate results in a 17 unit mass decrease of theparent peptide. The subsequent hydrolysis results in an 18 unit massincrease. Isolation of the succinimide intermediate is difficult due toinstability under aqueous conditions. As such, deamidation is typicallydetectable as 1 unit mass increase. Deamidation of an asparagine resultsin either aspartic or isoaspartic acid. The parameters affecting therate of deamidation include pH, temperature, solvent dielectricconstant, ionic strength, primary sequence, local polypeptideconformation and tertiary structure. The amino acid residues adjacent toAsn in the peptide chain affect deamidation rates. Gly and Ser followingan Asn in protein sequences results in a higher susceptibility todeamidation.

In certain embodiments, the liquid formulation for use in a method oftreatment described in the present disclosure may be preserved underconditions of pH and humidity to prevent deamidation of the proteinproduct.

The aqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation. Illustrativecarriers include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

Intravenous (IV) formulations may be the preferred administration routein particular instances, such as when a patient is in the hospital aftertransplantation receiving all drugs via the IV route. In certainembodiments, the liquid formulation is diluted with 0.9% Sodium Chloridesolution before administration. In certain embodiments, the diluted drugproduct for injection is isotonic and suitable for administration byintravenous infusion.

In certain embodiments, a salt or buffer component may be added in anamount of 10 mM-200 mM. The salts and/or buffers are pharmaceuticallyacceptable and are derived from various known acids (inorganic andorganic) with “base forming” metals or amines. In certain embodiments,the buffer may be phosphate buffer. In certain embodiments, the buffermay be glycinate, carbonate, citrate buffers, in which case, sodium,potassium or ammonium ions can serve as counterion.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

The aqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation. Illustrativecarriers include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

In another aspect the present disclosure also provides sustained releaseformulations (long-lasting formulation) suitable for injection todeliver a comprising Cardiac-specific Targeting-Peptide (CTP) conjugatedto Neuregulin-1β or a moiety having antiarrhythmic properties.

VII. Dosage Regimen

In one aspect, provided herein is sustained-delivery formulation of apeptide comprising a Cardiac-specific Targeting-Peptide (CTP) (e.g., anyone of SEQ ID NOs: 1-35) linked to a drug or therapeutic (e.g.,amiodarone). In certain embodiments, the formulation uses a controlledrelease system. In certain embodiments, the formulation uses a slowrelease system. In certain embodiments, a sustained-delivery formulationof the present disclosure includes a peptide comprising a CTP_(12aa)(SEQ ID NO: 1) linked to a therapeutic agent (e.g., amiodarone). Incertain embodiments, a sustained-delivery formulation of the presentdisclosure includes a peptide comprising a CTP_(12aa) (SEQ ID NO: 2)linked to a therapeutic agent (e.g., amiodarone). In certainembodiments, a sustained-delivery formulation of the present disclosureincludes a peptide comprising a CTP_(6aa) (SEQ ID NO: 5) linked to atherapeutic agent (e.g., amiodarone).

In certain embodiments, the formulation delivers a peptide comprising aCardiac-specific Targeting-Peptide (CTP) (e.g., any one of SEQ ID NOs:1-35) of the present disclosure linked to a therapeutic agent (e.g.,amiodarone) over a period of at least 6 hours (e.g., 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours). In certain embodiments, the formulationdelivers a peptide comprising a CTP_(12aa) represented by SEQ ID NO: 1linked to a drug or therapeutic (e.g., amiodarone) over a period of atleast 6 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours). Incertain embodiments, the formulation delivers a peptide comprising aCTP_(12aa) represented by SEQ ID NO: 2 linked to a drug or therapeutic(e.g., amiodarone) over a period of at least 6 hours (e.g., 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 24 hours). In certain embodiments, theformulation delivers a peptide comprising a CTP_(6aa) represented by SEQID NO: 5 linked to a drug or therapeutic (e.g., amiodarone) over aperiod of at least 6 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24hours).

In certain embodiments, the formulation delivers a peptide of thepresent disclosure (e.g., any one of SEQ ID NOs: 1-35) linked to atherapeutic agent (e.g., amiodarone) over a period of 6 hours, 7 hours,8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, or 24 hours. In certain embodiments, the formulationdelivers a peptide comprising a CTP_(12aa) represented by SEQ ID NO: 1linked to a drug or therapeutic (e.g., amiodarone) over a period of 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, or 24 hours. In certainembodiments, the formulation delivers a peptide comprising a CTP_(12aa)represented by SEQ ID NO: 2 linked to a drug or therapeutic (e.g.,amiodarone) over a period of 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24hours. In certain embodiments, the formulation delivers a peptidecomprising a CTP_(6aa) represented by SEQ ID NO: 5 linked to a drug ortherapeutic (e.g., amiodarone) over a period of 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, or 24 hours.

In certain embodiments, the formulation delivers a peptide of thepresent disclosure (e.g., any one of SEQ ID NOs: 1-35) linked to atherapeutic agent (e.g., amiodarone) at least once daily (e.g., once aday, twice a day, three times a day). In certain embodiments, theformulation delivers a peptide comprising a CTP_(12aa) represented bySEQ ID NO: 1 linked to a drug or therapeutic (e.g., amiodarone) at leastonce daily (e.g., once a day, twice a day, three times a day). Incertain embodiments, the formulation delivers a peptide comprising aCTP_(12aa) represented by SEQ ID NO: 2 linked to a drug or therapeutic(e.g., amiodarone) at least once daily (e.g., once a day, twice a day,three times a day). In certain embodiments, the formulation delivers apeptide comprising a CTP_(6aa) represented by SEQ ID NO: 5 linked to adrug or therapeutic (e.g., amiodarone) at least once daily (e.g., once aday, twice a day, three times a day).

In certain embodiments, the formulation delivering a peptide of thepresent disclosure (e.g., any one of SEQ ID NOs: 1-35) linked to atherapeutic agent (e.g., amiodarone) is administered at least once aweek (e.g., once a week, twice a week, 3, 4, 5, 6, 7, or more times aweek). In certain embodiments, the formulation delivering a peptidecomprising a CTP_(12aa) represented by SEQ ID NO: 1 linked to a drug ortherapeutic (e.g., amiodarone) is administered at least once a week(e.g., once a week, twice a week, 3, 4, 5, 6, 7, or more times a week).In certain embodiments, the formulation delivering a peptide comprisinga CTP_(12aa) represented by SEQ ID NO: 2 linked to a drug or therapeutic(e.g., amiodarone) is administered at least once a week (e.g., once aweek, twice a week, 3, 4, 5, 6, 7, or more times a week). In certainembodiments, the formulation delivering a peptide comprising a CTP_(6aa)represented by SEQ ID NO: 5 linked to a drug or therapeutic (e.g.,amiodarone) is administered at least once a week (e.g., once a week,twice a week, 3, 4, 5, 6, 7, or more times a week).

In some embodiments, the formulation delivering a peptide of the presentdisclosure (e.g., any one of SEQ ID NOs: 1-35) linked to a therapeuticagent (e.g., amiodarone) is a sustained release formulation administeredabout once per month, about once per every 2 months, about once perevery 3 months, about once per every 2 weeks, about once per every 3weeks, about once per every 4 weeks, about once per every 5 weeks, aboutonce per every 6 weeks, about once per every 7 weeks, about once perevery 8 weeks, about once per every 9 weeks, or about once per every 10weeks. In certain embodiments, the formulation delivering a peptidecomprising a CTP_(12aa) represented by SEQ ID NO: 1 linked to a drug ortherapeutic (e.g., amiodarone) is a sustained release formulationadministered about once per month, about once per every 2 months, aboutonce per every 3 months, about once per every 2 weeks, about once perevery 3 weeks, about once per every 4 weeks, about once per every 5weeks, about once per every 6 weeks, about once per every 7 weeks, aboutonce per every 8 weeks, about once per every 9 weeks, or about once perevery 10 weeks. In certain embodiments, the formulation delivering apeptide comprising a CTP_(12aa) represented by SEQ ID NO: 2 linked to adrug or therapeutic (e.g., amiodarone) is a sustained releaseformulation administered about once per month, about once per every 2months, about once per every 3 months, about once per every 2 weeks,about once per every 3 weeks, about once per every 4 weeks, about onceper every 5 weeks, about once per every 6 weeks, about once per every 7weeks, about once per every 8 weeks, about once per every 9 weeks, orabout once per every 10 weeks. In certain embodiments, the formulationdelivering a peptide comprising a CTP_(6aa) represented by SEQ ID NO: 5linked to a drug or therapeutic (e.g., amiodarone) is a sustainedrelease formulation administered about once per month, about once perevery 2 months, about once per every 3 months, about once per every 2weeks, about once per every 3 weeks, about once per every 4 weeks, aboutonce per every 5 weeks, about once per every 6 weeks, about once perevery 7 weeks, about once per every 8 weeks, about once per every 9weeks, or about once per every 10 weeks.

In some embodiments, a peptide of the present disclosure (e.g., any oneof SEQ ID NOs: 1-35) linked to a therapeutic agent (e.g., amiodarone orNeuregulin-1β) is administered at a rate of about 100 mg, 200 mg, 300mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg,1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg,or 2000 mg over a period of 24 hours, for example, the first 24 hourperiod of administration. In some embodiments, a formulation describedherein is administered at a rate of between 100 mg and 500 mg, between100 mg and 1000 mg, between 500 mg and 1000 mg, between 700 mg and 1000mg, between 700 mg and 1500 mg, between 1200 mg and 1500 mg, between1000 mg and 1500 mg, between 1000 mg and 2000 mg, between 1500 mg and2000 mg, between 1700 mg and 2000 mg, between 800 mg and 1200 mg,between 800 and 1000 mg, between 800 mg and 1600 mg, between 600 mg and800 mg, between 400 mg and 600 mg, or between 900 and 1200 mg over aperiod of 24 hours, for example, the first 24 hour period ofadministration. In some embodiments, a peptide of the present disclosure(e.g., any one of SEQ ID NOs: 1-35) linked to a therapeutic agent (e.g.,amiodarone or Neuregulin-1β) is administered in the form of aformulation that is administered orally, intravenously, enterally,parenterally, topically, by injection, intramuscularly, intraarterially,intrathecally, intracapsularly, intraorbitally, intracardiacly,intradermally, intraperitoneally, transtracheally, subcutaneously,subcuticularly, intraarticularly, subcapsularly, subarachnoidally,intraspinally, by epidural or infrasternal injection, or by infusion.

In some embodiments, a formulation described herein can be administeredby an infusion protocol. For example, in some embodiments, a formulationcan be administered, for example, by infusion, for example, in the formof a saline solution, where the solution includes a peptide of thepresent disclosure (e.g., any one of SEQ ID NOs: 1-35) linked to atherapeutic agent (e.g., amiodarone or Neuregulin-1β). In someembodiments the peptide linked to a therapeutic agent is administered toa patient in an amount of about 25 mg, 50 mg, 75 mg, 100 mg, 110 mg, 125mg, 130 mg, 140 mg, 150 mg, 160 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, 400 mg, 450 mg, 500 mg, 600mg, 700 mg, 720 mg, 750 mg, 0 mg to 100 mg, 50 mg to 150 mg, 50 mg, to100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 200 mg to 400 mg, 200 mg to500 mg, 100 mg to 150 mg, 100 mg to 500 mg, 150 mg to 300 mg, 100 mg to360 mg, 100 mg to 400 mg, 300 mg to 500 mg, 300 mg to 600 mg, 300 mg to600 mg, or 300 mg to 700 mg over a period of about 5 minutes, 10minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours 9hours 10 hours, 11 hours, 12 hours, 13 hours, 14 hours 15 hours 16 hours17 hours, 18 hours, 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 1week.

In some embodiments, a formulation described herein can be administeredby an infusion protocol. For example, in some embodiments, a formulationcan be administered, for example, by infusion, for example, in the formof a saline solution, where the solution includes a peptide of thepresent disclosure (e.g., any one of SEQ ID NOs: 1-35) linked to atherapeutic agent (e.g., amiodarone or Neuregulin-1β). In someembodiments the peptide linked to a therapeutic agent is administered toa patient in an amount of about 25 mg, 50 mg, 75 mg, 100 mg, 110 mg, 125mg, 130 mg, 140 mg, 150 mg, 160 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 0 mg to 100 mg, 50 mg to 150mg, 50 mg, to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 200 mg to 400mg, 200 mg to 500 mg, 100 mg to 150 mg, 100 mg to 500 mg, or 150 mg to300 mg over about the first 1 minute, 3 minutes, 5 minutes, 7 minutes,10 minutes, 12 minutes, 15 minutes, 20 minutes, or 30 minutes ofinfusion; after which the peptide linked to the therapeutic agent isadministered to the patient in an amount of about 25 mg, 50 mg, 75 mg,100 mg, 110 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 175 mg, 200 mg,225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, 400 mg,450 mg, 500 mg, 600 mg, 700 mg, 720 mg, 750 mg, 0 mg to 100 mg, 50 mg to150 mg, 50 mg, to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 200 mg to400 mg, 200 mg to 500 mg, 100 mg to 150 mg, 100 mg to 500 mg, 150 mg to300 mg, 100 mg to 360 mg, 100 mg to 400 mg, 300 mg to 500 mg, 300 mg to600 mg, 300 mg to 600 mg, or 300 mg to 700 mg over the next 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours of infusion;after which the peptide linked to the therapeutic agent is administeredto the patient in an amount of about 25 mg, 50 mg, 75 mg, 100 mg, 110mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 175 mg, 200 mg, 225 mg, 250mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, 400 mg, 450 mg, 475mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580mg, 590 mg, 600 mg, 700 mg, 720 mg, 750 mg, 800 mg, 900 mg, 950 mg, 1000mg, 1050 mg, 1080 mg, 0 mg to 100 mg, 50 mg to 150 mg, 50 mg, to 100 mg,100 mg to 200 mg, 200 mg to 300 mg, 200 mg to 400 mg, 200 mg to 500 mg,100 mg to 150 mg, 100 mg to 500 mg, 150 mg to 300 mg, 100 mg to 360 mg,100 mg to 400 mg, 300 mg to 500 mg, 300 mg to 600 mg, 300 mg to 600 mg,or 300 mg to 700 mg, 100 mg to 550 mg, 200 mg to 550 mg, 300 mg to 550mg, 400 mg to 550 mg, 500 mg to 600 mg, 500 mg to 700 mg, 500 mg to 800mg, 500 mg to 900 mg, 500 mg to 1000 mg, 500 mg to 1100 mg, or 400 mg to1100 mg over the next 30 minutes, 1 hour, 2 hours 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, or 36 hours ofinfusion. In a particular embodiment, the peptide linked to thetherapeutic agent is administered to the patient in an amount of about150 mg over the first 10 minutes of infusion, followed by 360 mg overthe next 6 hours, and followed by 540 mg over the next 18 hours.

In some embodiments, a formulation described herein is administered, forexample, by infusion, where a peptide described herein (e.g., any one ofSEQ ID NOs: 1-35) linked to a therapeutic agent (e.g., amiodarone orNeuregulin-1β) is administered at a specified rate after about the first24 hours of administration. For example, in some embodiments describedherein, after the first 24 hours of administration, the peptide linkedto a therapeutic agent is administered to a patient in an amount ofabout 25 mg, 50 mg, 75 mg, 100 mg, 110 mg, 125 mg, 130 mg, 140 mg, 150mg, 160 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350mg, 360 mg, 375 mg, 400 mg, 450 mg, 475 mg, 500 mg, 510 mg, 520 mg, 530mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 700 mg, 720mg, 750 mg, 800 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1080 mg, 1100 mg,1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 0 mg to 100 mg, 50 mg to150 mg, 50 mg, to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 200 mg to400 mg, 200 mg to 500 mg, 100 mg to 150 mg, 100 mg to 500 mg, 100 mg to600 mg, 150 mg to 300 mg, 100 mg to 360 mg, 100 mg to 400 mg, 300 mg to500 mg, 300 mg to 600 mg, 300 mg to 600 mg, or 300 mg to 700 mg, 100 mgto 550 mg, 200 mg to 550 mg, 300 mg to 550 mg, 400 mg to 550 mg, 400 mgto 600 mg, 500 mg to 600 mg, 500 mg to 700 mg, 500 mg to 800 mg, 500 mgto 900 mg, 500 mg to 1000 mg, 500 mg to 1100 mg, 600 mg to 800 mg, 600mg to 1000 mg, 1000 mg to 1500 mg, 1000 mg to 1200 mg, 1000 mg to 1400mg, 1100 mg to 1500 mg, or 400 mg to 1100 mg over 30 minutes, 1 hour, 2hours 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24hours, 27 hours, 30 hours, or 36 hours following the first 24 hours ofadministration. in a particular embodiment described herein, after thefirst 24 hours of administration, the peptide linked to a therapeuticagent is administered to a patient in an amount of about 720 mg over 24hours following the first 24 hours of administration. In embodimentsdescribed herein, the aforementioned administration may be continued fora period of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week,10 days, 12 days, 2 weeks, 15 days, 18 days, 20 days, 3 weeks, 4 weeks,5 weeks, 6 weeks, 7 weeks, 8 weeks 9 weeks, 10 weeks, 11 weeks, 12weeks, or longer.

In some embodiments, a formulation described herein is administered, forexample, by infusion or subcutaneously, where a peptide described herein(e.g., any one of SEQ ID NOs: 1-35) linked to a therapeutic agent (e.g.,amiodarone) is administered to achieve a serum circulating level of thetherapeutic of about 10-2500 ng/mL in a patient. For example, in someembodiments described herein, after the first 24 hours ofadministration, the peptide linked to amiodarone is administered to apatient in an amount to achieve a serum circulating level of about 10ng/mL, 25 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 110 ng/mL, 125 ng/mL,130 ng/mL, 140 ng/mL, 150 ng/mL, 160 ng/mL, 175 ng/mL, 200 ng/mL, 225ng/mL, 250 ng/mL, 275 ng/mL, 300 ng/mL, 325 ng/mL, 350 ng/mL, 360 ng/mL,375 ng/mL, 400 ng/mL, 450 ng/mL, 475 ng/mL, 500 ng/mL, 510 ng/mL, 520ng/mL, 530 ng/mL, 540 ng/mL, 550 ng/mL, 560 ng/mL, 570 ng/mL, 580 ng/mL,590 ng/mL, 600 ng/mL, 700 ng/mL, 720 ng/mL, 750 ng/mL, 800 ng/mL, 900ng/mL, 950 ng/mL, 1000 ng/mL, 1050 ng/mL, 1080 ng/mL, 1100 ng/mL, 1200ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 10 ng/mL to 100ng/mL, 50 ng/mL to 150 ng/mL, 50 ng/mL to 100 ng/mL, 100 ng/mL to 200ng/mL, 200 ng/mL to 300 ng/mL, 200 ng/mL to 400 ng/mL, 200 ng/mL to 500ng/mL, 100 ng/mL to 150 ng/mL, 100 ng/mL to 500 ng/mL, 100 ng/mL to 600ng/mL, 150 ng/mL to 300 ng/mL, 100 ng/mL to 360 ng/mL, 100 ng/mL to 400ng/mL, 300 ng/mL to 500 ng/mL, 300 ng/mL to 600 ng/mL, 300 ng/mL to 600ng/mL, or 300 ng/mL to 700 ng/mL, 100 ng/mL to 550 ng/mL, 200 ng/mL to550 ng/mL, 300 ng/mL to 550 ng/mL, 400 ng/mL to 550 ng/mL, 400 ng/mL to600 ng/mL, 500 ng/mL to 600 ng/mL, 500 ng/mL to 700 ng/mL, 500 ng/mL to800 ng/mL, 500 ng/mL to 900 ng/mL, 500 ng/mL to 1000 ng/mL, 500 ng/mL to1100 ng/mL, 600 ng/mL to 800 ng/mL, 600 ng/mL to 1000 ng/mL, 1000 ng/mLto 1500 ng/mL, 1000 ng/mL to 1200 ng/mL, 1000 ng/mL to 1400 ng/mL, 1100ng/mL to 1500 ng/mL, or 400 ng/mL to 1100 ng/mL over 30 minutes, 1 hour,2 hours 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24hours, 27 hours, 30 hours, or 36 hours following the first 24 hours ofadministration. In a particular embodiment described herein, the peptideof SEQ ID NO: 1, 2, or 5 linked to amiodarone is administered to apatient in an amount to achieve a serum circulating level of amiodaroneof about 10 to 2500 ng/mL for 24 hours to 7 days following the first 24hours of administration. In embodiments described herein, theaforementioned administration may be continued for a period of about 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 12 days, 2weeks, 15 days, 18 days, 20 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer.

VIII. Definitions

The term “CTP” as used herein refers to a Cardiac-specificTargeting-Peptide. In certain embodiments, the CTP is comprised of a6-amino acid long peptide, CTP_(6aa). In certain embodiments, theCTP_(12aa) is comprised of a 12-amino acid long peptide. In certainembodiments, the CTP is conjugated to a moiety having antiarrhythmicproperties.

The term “effective amount” as used herein refers to the amount of anactive agent (e.g., a CTP of any one of SEQ ID NOs: 1-35, for exampleSEQ ID NO: 1, 2, or 5, of the present disclosure) sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages and is notintended to be limited to a particular formulation or administrationroute.

As used herein, “treat, “treating,” and “treatment” mean the treatmentof a disease in a subject, e.g., in a human. This includes: (a)inhibiting the disease, i.e., arresting its development; and (b)relieving the disease, i.e., causing regression of the disease state. Asused herein, the terms “subject” and “patient” refer to an organism tobe treated by the methods and compositions described herein. Suchorganisms preferably include, but are not limited to, mammals (e.g.,murines, simians, equines, bovines, porcines, canines, felines, and thelike), and more preferably includes humans.

The methods and compositions described herein can be used alone or incombination with other therapeutic agents and/or modalities. The termadministered “in combination,” as used herein, is understood to meanthat two (or more) different treatments are delivered to the subjectduring the course of the subject's affliction with the disorder, suchthat the effects of the treatments on the patient overlap at a point intime. In certain embodiments, the delivery of one treatment is stilloccurring when the delivery of the second begins, so that there isoverlap in terms of administration. This is sometimes referred to hereinas “simultaneous” or “concurrent delivery.” In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In certain embodiments of either case, the treatmentis more effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In certainembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present disclosure that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present disclosure that consist essentially of, or consist of,the recited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentdisclosure, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present disclosure and/or inmethods of the present disclosure, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent disclosure also includes the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present disclosure remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present disclosure and does not pose a limitation on thescope of the invention unless claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the present disclosure.

EXAMPLES

The following Examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1—Alanine Scan Analysis of the Cardiac-Specific TargetingPeptide

This example describes the design and testing of cardiac-specifictargeting peptide variants to determine the contribution of each aminoacid residue to transduction activity.

Previous studies had identified a synthetic 12-amino acid peptide(APWHLSSQYSRT; SEQ ID NO: 1) capable of transducing cardiomyocytes.Alanine scanning analysis of the APWHLSSQYSRT (SEQ ID NO: 1) sequencewas performed to assess the importance and relative contribution of eachamino acid residue to the cell penetrating ability.

Variants of SEQ ID NO: 1 in which every amino acid was systematicallymutated to alanine were synthesized in the University of PittsburghPeptide Synthesis Facility. Each variant was conjugated to theCyanine5.5 fluorescent dye (Cy5.5). The rat cardiomyocyte cell line H9C2(ATCC, catalogue #: CRL-1446) was incubated for 30 minutes at 37° C.with Dimethyl sulfoxide (DMSO) as a control, or the alanine peptidevariants. Cells were then washed 3 times with PBS, trypsinized andresuspended, stained for live/dead cells, and then FACS sorted for Cy5.5positive cells.

The fluorescence intensities of H9C2 cells transduced with the testedalanine variants are shown in FIG. 1A and FIG. 1B. The percentage ofCy5.5 positive H9C2 cells, normalized to the non-mutated peptide (SEQ IDNO: 1), for each peptide variant is summarized in Table 2 below.

TABLE 2 SEQ ID Peptide Peptide % transduction NO Name Sequenceof H9C2 Cells DMSO Negative Control   1.87 1 CTP APWHLSSQYSRT 100 2 P2AAAWHLSSQYSRT  59.15 35 W3A APAHLSSQYSRT  75.81 26 H4A APWALSSQYSRT 55.05 27 LSA APWHASSQYSRT  87.81 28 S6A APWHLASQYSRT 107.21 29 S7AAPWHLSAQYSRT  54.32 30 Q8A APWHLSSAYSRT  67.35 31 Y9A APWHLSSQASRT 10732 S10A APWHLSSQYART  30.61 33 R11A APWHLSSQYSAT  32.75 34 T12AAPWHLSSQYSRA  90.33

TABLE 3 (data represented in FIG. 1C). Fluorescence of the variouspeptides measured using spectrophotometric data in order to normalizethe fluorescence activated cell sorting of H9C2 cells to control fordifferences in fluorescence between peptides. Sample Average CorrectionSD Correction No Treatment 201.7 2.5 Live-Dead Stain 217 1.7 CTP 5996329.2 P2A 9535.0 331.3 W3A 5332.5 64.4 H4A 7537.1 550.1 L5A 5323.2 225.9S6A 18868.9 1376.7 S7A 22813.0 1396.8 Q8A 9371.3 339.2 Y9A 5465.9 111.5S10A 3775.1 161.6 R11A 10255.7 254.4 T12A 15162.7 746.5 CTP-B 6124.6415.1

TABLE 4 (data represented in FIG. 1D): Fluorescence activated cellsorting of H9C2 cells after a 30 min incubation with 10 uM of variouspeptides. Raw Median Correction Corrected Median Sample Cy5.5 RatioCy5.5 No Treatment_001 202 N/a 202 No Treatment_002 199 N/a 199 NoTreatment_003 204 N/a 204 Live-Dead_001 216 N/a 216 Live-Dead_002 216N/a 216 Live-Dead_003 219 N/a 219 CTP_001 5652 1 5652.0 CTP_002 6028 16028.0 CTP_003 6308 1 6308.0 P2A_001 9449 0.95 9901.8 P2A_002 8834 0.959298.9 P2A_003 9014 0.95 9488.4 W3A_001 4862 0.91 5369.7 W3A_002 48620.91 5369.7 W3A_003 4761 0.91 5258.1 H4A_001 7173 0.94 7666.1 H4A_0027496 0.94 8011.3 H4A_003 6488 0.94 6934.0 L5A_001 3107 0.61 5077.5L5A_002 3379 0.61 5522.0 L5A_003 3286 0.61 5370.0 S6A_001 22342 1.2617793.0 S6A_002 23096 1.26 18393.5 S6A_003 25641 1.26 20420.3 S7A_00120184 0.95 21208.1 S7A_002 22342 0.95 23475.6 S7A_003 22608 0.95 23755.1Q8A_001 10041 1.03 9725.5 Q8A_002 9642 1.03 9339.0 Q8A_003 9343 1.039049.4 Y9A_001 5018 0.94 5365.7 Y9A_002 5093 0.94 5445.9 Y9A_003 52240.94 5586.0 S10A_001 1722 0.46 3707.6 S10A_002 1699 0.46 3658.1 S10A_0031839 0.46 3959.6 R11A_001 9555 0.93 10237.9 R11A_002 9343 0.93 10010.7R11A_003 9817 0.93 10518.6 T12A_001 18458 1.29 14307.2 T12A_002 202321.29 15682.3 T12A_003 19995 1.29 15498.6 CTP-B_001 4711 0.8317867275663.7 CTP-B_002 5381 0.831786727 6469.2 CTP-B_003 5191 0.8317867276240.8

These results demonstrated that amino acid residues in the C-terminus ofSEQ ID NO: 1 make a major contribution to the transduction ability ofthe peptide.

Example 2—In Vitro Transduction and Viability Studies of N- andC-Terminal Cardiac-Specific Targeting Peptides

The results described in Example 1 suggested that the cell penetratingability of the cardiac-specific targeting peptide (APWHLSSQYSRT; SEQ IDNO: 1) resides in the 6 C-terminal amino acid residues. To evaluate thisfurther, variants of SEQ ID NO: 1, including linear and cyclized 6-aminoacid N- and C-terminal variants, were synthesized in the University ofPittsburgh Peptide Synthesis Facility. Each variant was conjugated toCy5.5.

Solid-phase peptide synthesis were used to synthesize the full-lengthform (NH₂-APWHLSSQYSRT-COOH (SEQ ID NO: 1)), as well as an N-terminus 6amino acid called CTP-A (NH₂-APWHLS-COOH (SEQ ID NO: 24)), or asCTP_(6aa) (NH₂-SQYSRT-COOH (SEQ ID NO: 5)) peptides. Full length andCTP_(6aa) were also synthesized in a cyclic forms. A random, linear,12-amino acid long peptide (RAN: NH₂-STLMKFCYVEQN-COOH (SEQ ID NO: 26))was also synthesized to serve as control. All peptides werefluorescently labeled with Cyanine5.5 (Cy5.5). Isolated, beatingneonatal mouse cardiomyocytes (CMC) were incubated with 10 μM of thepeptides at 37° C. for 30 minutes, washed extensively with PBS andconfocal microscopy performed. Additionally, H9C2 cells, a ratcardiomyoblast cell line, were incubated with Dimethyl sulfoxide (DMSO)or the peptides labeled with Cy5.5 at 5 μM for 30 minutes at 37° C.,washed 3× with PBS, trypsinized and resuspended, stained for live/deadcells, and then FACS sorted for Cy5.5 positive cells.

As shown in FIG. 2, shorter, linear versions of the cardiac-specifictargeting peptide (APWHLSSQYSRT; SEQ ID NO: 1) were able to efficientlytransduce the three cell lines tested. Across all three cell lines, thelinear 6-amino acid C-terminal peptide variant transduced cells at leastas well as the full-length peptide (compare CTP_(6aa) to CTP in FIGS.2A-2D). Cyclization of the 6-amino acid peptide variants decreased thetransduction efficiency of H9C2 cells considerably (FIGS. 2A-2B).Compared to the full-length peptide and DMSO control, transduction ofH9C2 cells and MEFs with the linear 6 amino acid C-terminal peptidevariants did not alter cell viability (FIG. 2E).

These results demonstrated that the six C-terminal amino acid residues(SQYSRT; SEQ ID NO: 5) are sufficient to confer the cell penetratingability of the 12-amino acid, full-length cardiac-specific targetingpeptide (APWHLSSQYSRT; SEQ ID NO: 1).

Example 3—Delivery of Rhodamine Cargo to Cardiomyocytes Via Conjugationto the C-Terminus of the Cardiac-Specific Targeting Peptide

This example describes the use of the cardiac-specific targeting peptidevia conjugation at the C-terminus to deliver a rhodamine cargo to theintracellular compartments of cardiomyocytes.

The cardiac-specific targeting peptide was synthesized in the Universityof Pittsburgh Peptide Synthesis Facility and labeled at the N-terminuswith carboxyfluorescein (green), and with rhodamine (red) at theC-terminus through an ester linkage. Beating cardiomyocytes derived fromhuman induced pluripotent stem cells (iPSC) were then incubated with thedual labeled peptide for 30 minutes at 37° C., washed 3× with PBS andimmediately thereafter, live cell imaging was performed using confocalmicroscopy.

As shown in FIG. 3C, the dual labeled peptide successfully entered thebeating human iPSC derived cardiomyocytes, and also that ester-linkageof a cargo to the C-terminus of the cardiac-specific targeting peptideresulted in delivery of the cargo to intracellular compartments due tocleavage of the ester linkage by intracellular esterase(s). FIG. 3Cdemonstrates that the rhodamine (red) moiety conjugated to theC-terminus of CTP remained inside the cells while the carboxyfluorescein(green) moiety was expelled. Therefore, the data showed that conjugationthe rhodamine cargo to the C-terminus of the cardiac-specific targetingpeptide (e.g., CTP_(6aa)) delivered the cargo inside of cells, while thecargo conjugated to the N-terminus was ejected, which in effect, workedas an internal control.

These results demonstrated that the C-terminus of the cardiac-specifictargeting peptide, when conjugated via an ester linkage to a cargo, suchas rhodamine, successfully delivered the cargo internally tocardiomyocytes.

Example 4—Uptake of Cy5.5-Labeled CTP by Myocardial Tissue

In order to analyze the rate of uptake of labeled CTP peptides bymyocardial tissue, 6- to 8-week-old wild-type CD1 mice were injectedintravenously with a Cy5.5-labeled CTP (CTP-Cy5.5) or a control 12 aminoacid random peptide sequence labeled with Cy5.5 (RAN-Cy5.5) at a dose of10 mg/kg. The peptides were allowed to circulate for 15, 30, 45, 60, 90,or 360 min, after which mice were euthanized, fixed with 3 mL of 10%formalin, and heart tissue was dissected out. Tissue was fixed andembedded in paraffin, and sectioned. The sections were cross-stainedwith nuclear stain 40,6-diamidino-2-phenylindole (DAPI) and mounted, andfluorescent microscopy was performed. Fluorescence was quantified usingImageJ (National Institute of Health, Bethesda, Md., USA). Alltime-points and peptides were run in triplicate. Robust uptake ofCTP-Cy5.5 was observed, peaking at 15 minutes post injection (FIG. 5).Significantly more uptake of CTP-Cy5.5 was observed at each time pointcompared to uptake of RAN-Cy5.5.

Additionally, fluorescently-labeled CTP variants (CTP, CTP-B, H4A, P2A)or fluorescently-labeled random peptide sequence (RAN) were injectedinto mice and fluorescence intensity was measured in fixed myocardialtissue. Mice were injected with peptides, and, 15 minutes afterinjection, heart tissue was dissected, cryosectioned, stained, andanalyzed for fluorescence intensity. Results are shown in FIG. 6A andFIG. 6B. Results in FIG. 6B show that fluorescence intensity inmyocardial tissue was significantly greater after injection of CTP-B orP2A compared to CTP. These results demonstrate that CTP peptides aretaken up rapidly by myocardial tissue (reaching peak uptake 15 minutesafter injection) and that CTP-B and alanine peptide variants showsuperior uptake compared to random peptide sequence and CTP.

Example 5—Amiodarone-CTP for Treating Atrial and/or VentricularArrhythmias

Solid phase peptide synthesis (SPPS) of the Amiodarone-CTP peptides isperformed with synthesis of the CTP peptides on Rink Amide MBHA resinusing Fluorenylmethyloxycarbonyl (FMOC) chemistry with Oxyma/DIC(Ethyl-(2Z)-2-cyano-2-hydroxyiminoacetate/N,N Diisopropylcarbodiimide)activation on a Liberty CEM microwave synthesizer. After completion ofthe peptide chain assembly, the N-terminal amino group of theNH2-CTP-MBHA resin is conjugated with 6-(Fmoc-amino)-hexanoic acid inDIPEA/TBTU/Hobt/DMF overnight at room temperature. Next, the FMOC-groupis removed from the FMOC-hexanoyl-CTP peptides-MBHA resin using 20%Piperidine/DMF for 30 minutes at room temperature. Amiodarone-HCL (SigmaAldrich) then undergoes a microwave assisted coupling to the freeN-terminus of the NH2-hexanoyl-CTP peptides-MBHA resin through an iminelinkage (Schiff base). Cleavage of Amiodarone-hexanoyl-CTP peptides-MBHAresin is accomplished using TFA/scavengers for 2 hours at roomtemperature, and then precipitated with Diethyl Ether. Residualscavengers are extracted from the crude peptide by three rounds of etherwashes.

Samples are centrifuged to separate components. The resulting crudeAmiodarone-hexanoyl-CTP peptides-CONH2 peptide is then air dried andpurified by preparative C-5 RP-HPLC on a Waters Delta Prep 4000chromatography system using standard Acetonitrile/0.1% TFA gradientconditions. Analytical C-5 RP-HPLC characterization on a Waters Alliancechromatography system followed by MALDI-Tof analysis on an AppliedBiosystems Voyager workstation is used to confirm the expected purityand mass of the final product. Equilibration of the purifiedAmiodarone-hexanoyl-CTP peptides in aqueous solution is followed byanalytical reversed phase HPLC monitoring in order to verify thein-vitro release of Amiodarone via hydrolysis of the Schiff base.

Example 6—Neuregulin-1P-CTP for Treatment of SHF

Neuregulin-1β along with its receptors (ErbB2-4 is required for normalcardiac development and has been shown to reduce apoptosis, myocardialfibrosis and reduce infarct size in animal models of diabetes as well asinfarction. A few small clinical trials have also shown efficacy ofparenteral treatment with recombinant human Neuregulin-1β in patientswith SHF. However, progress in bringing this therapy to the clinic hasbeen hampered by significant liver toxicity as well as hypoglycemia.

This example provides a fusion protein of Neuregulin-1β and CTPpeptides, with CTP downstream of Neuregulin-1β or attached to itsC-terminus. Primers are designed for Neuregulin-1β with sequence of CTPadded to the reverse sequence. The combined fusion protein sequence isgenerated by PCR using mouse genomic DNA extracted from Lungs, Liver orKidneys. Standard cloning techniques are used to blunt-end ligate theproduct into a cloning T7 plasmid which is then used to transform BL21bacterial cells. Colonies that emerge carrying the relevant antibioticresistance are sequenced and the ones carrying the right sequence,expanded and preserved as the transformed cells of choice. For proteinproduction, these cells are grown in large scale (˜500-1000 ml) culturemedium and induced to produce the fusion protein underIsopropyl-β-D-thiogalactoside stimulation. The protein is isolated usingHis-tag and His-coated beads and characterized by Western blot.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent and scientific documentsreferred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A peptide comprising Cardiac-specificTargeting-Peptide (CTP) of the sequence of SEQ ID NO: 1, 2, 4, 5, 6, 7,8, 9, 10, 11, 28, 29, or 34 linked to a moiety having antiarrhythmicproperties.
 2. The peptide of claim 1, wherein the moiety is a class IIIantiarrhythmic compound or a class IV antiarrhythmic compound.
 3. Thepeptide of claim 1, wherein the moiety is amiodarone.
 4. The peptide ofclaim 1, wherein the CTP has sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
 5. 5. The peptideaccording to any one of claims 1-4, wherein the moiety havingantiarrhythmic properties is linked to the CTP using Schiff basechemistry.
 6. The peptide of any one of claims 1-5, wherein the moietyhaving antiarrhythmic properties is linked upstream of the N-terminus ofthe CTP peptide.
 7. The peptide of any one of claims 1-6, wherein thepeptide is optionally further connected to a label at the N- and/or theC-termini.
 8. The peptide of any one of claims 1-6, wherein the peptideis optionally further connected to a label at the C-termini.
 9. Thepeptide of claim 7 or 8, wherein the label is a green fluorescent moietyor a red fluorescent moiety.
 10. The peptide of claim 9, wherein thegreen florescent moiety is 6-carboxyfluorescein.
 11. The peptide ofclaim 9, wherein the red fluorescent moiety is Cy5.5.
 12. A formulationcomprising a peptide according to any one of claims 1-6 and apharmaceutically acceptable carrier.
 13. The formulation of claim 12 isa sustained-delivery formulation.
 14. The formulation of claim 12 or 13,wherein the formulation uses a controlled release system.
 15. Theformulation of claim 12 or 13, wherein the formulation uses a slowrelease system.
 16. The formulation of claim 12 or 13 delivers thepeptide according to any one of claims 1-6 over a period of at least 6hours.
 17. The formulation of any one of claims 12-16, wherein thedelivery is over a period of at least 12 hours.
 18. The formulation ofany one of claims 12-16, wherein the delivery is over a period of atleast 24 hours.
 19. The formulation of any one of claims 12-16, whereinthe formulation is administered at least once daily.
 20. The formulationof any one of claims 12-16, wherein the formulation is administered atleast once a week, once every two weeks, once a month, once every 30days, once every 45 days, or once every 60 days.
 21. A method ofintroducing a moiety having antiarrhythmic properties into a cardiacmuscle cell comprising administering to the cardiac muscle cell thepeptide of any one of claims 1-6 or a formulation of any one of claims12-20 in an amount effective to introduce the moiety havingantiarrhythmic properties into the muscle cell.
 22. The method of claim21, wherein the peptide further comprises a microsphere or nanoparticle.23. A method of treating a human subject suffering from atrial and/orventricular arrhythmias, comprising administering to the human subjectthe peptide of any one of claims 1-6 or a formulation comprising apeptide according to any one of claims 1-5 according to any one ofclaims 12-20.
 24. The method of claim 23, wherein the ventriculararrhythmias is selected from premature ventricular contractions,ventricular tachycardia, and ventricular fibrillation.
 25. The method ofclaim 23, wherein the atrial arrhythmias is atrial fibrillation.
 26. Asix amino acid Cardiac-specific Targeting-Peptide (CTP_(6aa)) comprisingthe sequence of Xaa₁ Xaa₂ Y Xaa₃ Xaa₄ T (SEQ ID NO: 4).
 27. TheCTP_(6aa) of claim 26 comprising the sequence of S Q Xaa₁ S R Xaa₂ (SEQID NO: 6).
 28. The CTP_(6aa) of claim 26, wherein Xaa₁ is serine (S).29. The CTP_(6aa) of claim 26, wherein Xaa₂ is glutamine (Q).
 30. TheCTP_(6aa) of claim 26, wherein Xaa₃ is serine (S).
 31. The CTP_(6aa) ofclaim 26, wherein Xaa₄ is arginine (R).
 32. The CTP_(6aa) of claim 26,wherein Xaa₁ and Xaa₂ are serine (S) and glutamine (Q), respectively.33. The CTP_(6aa) of claim 26, wherein Xaa₁ and Xaa₃ are both serine(S).
 34. The CTP_(6aa) of claim 26, wherein Xaa₁ and Xaa₄ are serine (S)and arginine (R), respectively.
 35. The CTP_(6aa) of claim 26, whereinXaa₂ and Xaa₃ are glutamine (Q) and serine (S), respectively.
 36. TheCTP_(6aa) of claim 26, wherein the CTP_(6aa) comprises the sequenceSQYSRT (SEQ ID NO: 5).
 37. The CTP_(6aa) of claim 27, wherein Xaa₁ isalanine (A) and the CTP_(6aa) comprises the sequence of SQASRXaa₂ (SEQID NO: 7), or optionally, Xaa₁ is tryptophan (W) and the CTP_(6aa)comprises the sequence of SQWSRXaa₂ (SEQ ID NO: 8), or Xaa₁ is tyrosine(Y) and the CTP_(6aa) comprises the sequence of SQYSRXaa₂ (SEQ ID NO:9).
 38. The CTP_(6aa) of claim 27, wherein Xaa₂ is threonine (T), andXaa₁ is alanine (A), tryptophan (W), or tyrosine (Y) comprising thesequence of SQASRT (SEQ ID NO: 10), SQWSRT (SEQ ID NO: 11), or SQYSRT(SEQ ID NO: 5), respectively.
 39. The CTP_(6aa) of claim 27, whereinXaa₂ is alanine (A).
 40. The CTP_(6aa) of claim 27, wherein Xaa₁ istyrosine (Y) and Xaa₂ is alanine (A).
 41. The CTP_(6aa) of claim 27,wherein the CTP_(6aa) comprises the sequence SQYSRT (SEQ ID NO: 5). 42.The CTP_(6aa) of any one of claims 26-41, wherein the CTP_(6aa) isconjugated or fused to a nanoparticle or a moiety having antiarrhythmicproperties.
 43. A method of treating cardiac tissue or a cardiaccondition in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a composition comprisingthe conjugated or fused CTP_(6aa) of claim
 42. 44. The CTP_(6aa) of anyone of claims 26-41, wherein the CTP_(6aa) is conjugated or fused to atafazzin peptide.
 45. The CTP_(6aa) of claim 44, wherein the CTP_(6aa)is conjugated or fused to the tafazzin peptide through a polypeptidelinker.
 46. A method of treating a subject having a disorder associatedwith a tafazzin deficiency or a remodeled cardiolipin deficiency,comprising administering to the subject a therapeutically effectiveamount of a composition comprising the tafazzin-conjugated CTP_(6aa) ofclaim 44 or
 45. 47. A method of treating a subject having, or at risk ofdeveloping Barth syndrome, comprising administering to the subject atherapeutically effective amount of a composition comprising thetafazzin-conjugated CTP_(6aa) of claim 44 or
 45. 48. The CTP_(6aa) ofany one of claims 26-41, wherein the CTP_(6aa) is conjugated to adetectable agent.
 49. The CTP_(6aa) of claim 48, wherein the detectableagent is a radionuclide or radioactive moiety, biotin, luciferase, anenzyme, rhodamine, a fluorophore, nanoparticle, microbubbles, liposomesor a luminescent moiety.
 50. The CTP_(6aa) of any one of claims 26-41formulated as a delivery vehicle/agent.
 51. The CTP_(6aa) of claim 50,wherein the CTP_(6aa) is conjugated to a drug or therapeutic, ananoparticle, a peptide, a protein, or a detectable agent.
 52. TheCTP_(6aa) of claim 51, wherein the CTP_(6aa) is conjugated to the drugor therapeutic, the nanoparticle, the peptide, the protein, or thedetectable agent via an ester linkage, disulfide or protease sensitivelinkers.
 53. The CTP_(6aa) of claim 51 or 52, wherein the nanoparticlecomprises a drug or therapeutic.
 54. The CTP_(6aa) of any one of claims51-53, wherein the drug or therapeutic is selected from an antibody orfragment thereof, SB239063, superoxide dismutase, HGF, FGF-1, FGF-2, anNF-κB inhibitor, NSD peptide, heme oxygenase, an antioxidant, iNOS,S100A1, catalase, glutathione peroxidase, a TGFβ inhibitor, VEGF, sonichedgehog protein, HGF, an IAP, prostaglandin, Pyrvinium Pamoate,Diprotin A, Szeto-Schiller peptide, cyclosporine, and amiodarone.
 55. Amethod of treating cardiac tissue or a cardiac condition in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a composition comprising the CTP_(6aa) of claim 54.56. A method of introducing a cargo into a cardiac muscle cellcomprising administering, to the cardiac muscle cell, an amount of acomplex comprising the CTP_(6aa) of any one of claims 26-41 linked to acargo effective to introduce the cargo into the muscle cell.
 57. Themethod of claim 56, wherein the cargo comprises a radioisotope,fluorescent marker, gadolinium marker, luciferase marker, microsphere ornanoparticle.
 58. A method of treating a human subject suffering from amyocardial infarction, comprising introducing a cargo into a cardiacmuscle cell of the human subject comprising administering, to the humansubject, a therapeutically effective amount of a complex comprising theCTP_(6aa) of any one of claims 26-41 linked to a cargo, where the cargoinhibits cell death, inhibits arrhythmias, improves contractility,lengthens subject survival, or a combination thereof.
 59. The method ofclaim 58, wherein the cargo is selected from an NF-κB inhibitor, NSDpeptide, heme oxygenase, an antioxidant, iNOS, S100A1, superoxidedismutase, catalase, glutathione peroxidase, a TGFβ inhibitor, VEGF,FGF-1, FGF-2, sonic hedgehog protein, HGF, a class III antiarrhythmiccompound, a class IV antiarrhythmic compound, and an IAP.
 60. A methodof treating a human subject suffering from a myocardial infarction,comprising introducing a cargo into a cardiac tissue of the humansubject comprising administering, to the human subject, atherapeutically effective amount of a complex comprising the CTP_(6aa)of any one of claims 26-41 linked to a cargo, wherein the cargo inhibitscell death, inhibits arrhythmias, improves contractility, lengthenssubject survival, or a combination thereof.
 61. The method of claim 60,wherein the cargo is selected from an NF-κB inhibitor, NBD peptide, hemeoxygenase, an antioxidant, iNOS, S100A1, superoxide dismutase, catalase,glutathione peroxidase, a TGFβ inhibitor, VEGF, FGF-1, FGF-2, sonichedgehog protein, HGF, a class III antiarrhythmic compound, a class IVantiarrhythmic compound, and an IAP.
 62. A method of treating a subjectsuffering from a metabolic defect that damages the heart comprisingintroducing a cargo into a cardiac muscle cell of the human subjectcomprising administering, to the subject, a therapeutically effectiveamount of a complex comprising the CTP_(6aa) of any one of claims 26-41linked to a cargo, wherein the cargo corrects the metabolic defect. 63.The method of claim 62, wherein the metabolic defect is Gaucher'sdisease and the cargo is glucocerebrosidase.
 64. A method of treating asubject suffering from a metabolic defect that damages the heartcomprising introducing a cargo into a cardiac tissue of the humansubject comprising administering, to the subject, a therapeuticallyeffective amount of a complex comprising the CTP_(6aa) of any one ofclaims 26-41 linked to a cargo, where the cargo corrects the metabolicdefect.
 65. The method of claim 64, where the metabolic defect isGaucher's disease and the cargo is glucocerebrosidase.
 66. A peptidecomprising Cardiac-specific Targeting-Peptide (CTP) of the sequence ofSEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 28, 29, or 34 linked to an SSpeptide.
 67. The peptide according to claim 66, wherein the SS peptideis linked upstream of the N-terminus of the CTP peptide.
 68. The peptideaccording to claim 67, comprising an ester linkage between the CTP andthe SS peptide.
 69. The peptide according to claim 68, wherein the esterlinkage is an ester linkage cleavable by an intracellular esterase. 70.A peptide comprising Cardiac-specific Targeting-Peptide (CTP) of thesequence of SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 28, 29, or 34linked to heme-oxygenase 1 protein.
 71. The peptide of claim 70, whereinthe heme-oxygenase 1 protein is linked upstream of the N-terminus of theCTP peptide with an ester linkage.
 72. A peptide comprisingCardiac-specific Targeting-Peptide (CTP) of the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 28, 29, or 34 linked to a small moleculeselected from resveratrol, N-acetyl-cysteine,N-tert-butyl-α-phenylnitrone, and4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.
 73. A method of treatinga human subject suffering from a diastolic heart failure (DHF),comprising introducing a cargo into a cardiac muscle cell of the humansubject by administering to the human subject the peptide of any one ofclaims 66-69, wherein the SS peptide scavenges H₂O₂, and ONOO—, and/orLDL oxidation.
 74. The method of claim 73, wherein the cargo is selectedfrom the group consisting of SS-02 and SS-031.
 75. A method of treatingaging and/or oxidative stress related disease and/or disorder in asubject in thereof, comprising introducing the peptide of any one ofclaims 66-69 into an organ or a tissue of the subject.
 76. The method ofclaim 75, wherein the disease and/or disorder is selected fromischemia-reperfusion injury, neurodegenerative disease, diabetes,inflammatory diseases such as atherosclerosis, arthritis, and hepatitis.77. The peptide of any one of claims 66-72 for use in a method oftreating a subject by inhibiting mitochondrial permeability transition(MPT), preventing mitochondrial swelling, and/or reducing cytochrome crelease in response to Ca²⁺ overload.
 78. The peptide of any one ofclaims 66-72 for use in a method of treating a subject by minimizingmitochondrial permeability transition (MPT)-induced ROS accumulation,thereby reducing oxidative damage on mitochondria.
 79. A method oftreating a subject suffering from a metabolic defect that damages theheart, comprising introducing a peptide with ROS scavenging propertiesinto a cardiac muscle cell of the subject, comprising administering, tothe subject, the CTP linked to an SS peptide of any one of claims 66-69,wherein the SS peptide corrects the metabolic defect.
 80. The method ofclaim 79, wherein the metabolic defect is Gaucher's disease.